Novel gold nanostructures and methods of use

ABSTRACT

The invention is drawn to novel nanostructures comprising hollow nanospheres and nanotubes for use as chemical sensors, conduits for fluids, and electronic conductors. The nanostructures can be used in microfluidic devices, for transporting fluids between devices and structures in analytical devices, for conducting electrical currents between devices and structure in analytical devices, and for conducting electrical currents between biological molecules and electronic devices, such as bio-microchips.

The present application claims priority to U.S. Provisional PatentApplication Ser. No. 60/790,317 entitled “Synthesis Of Hollow GoldNanoshells And Methods Of Use”, filed Apr. 7, 2006, which is hereinincorporated by reference in its entirety for all purposes.

This invention was made partly using funds from the National ScienceFoundation, the Petroleum Research Fund/American Chemical Society, theUniversity of California at Santa Cruz, the student employee graduateresearch fellowship at Lawrence Livermore National Laboratory. This workwas performed under the auspices of the U.S. Department of Energy byUniversity of California Lawrence Livermore National Laboratory undercontract No. W-7405-Eng-48. The US Federal Government may have certainrights to this invention.

FIELD OF THE INVENTION

The present invention relates to structures comprising a metal withuseful properties. The structures are hollow spheres and tubes. Theinvention further relates to methods of using the structures fordetecting chemical and biological analytes and use in electronics and inmicrofluidics.

BACKGROUND

During the 1980s Raman Scattering in fibers was demonstrated by Lin,Stolen, and other co-workers of AT&T Bell Laboratories in Holmdel, N.J.,using lasers operating between 0.3 μm to 2.0 μm. In the early years ofthe Raman fiber before extensive work had begun, no one perceived that aRaman fiber could be pumped by a practical semiconductor laser-basedsource or that an efficient CW-pumped Raman Fiber Laser was possible.However, with the development of Cladding-pumped Fiber Lasers and FiberBragg Gratings, diode-laser-based CW Raman Fiber Lasers have been madeefficient, emitting at various wavelengths throughout the infraredspectrum a reality. (See van Gisbergen et al., (1996) Chem. Phys. Lett.259: 599-604.)

Raman spectroscopy is a powerful optical technique for detecting andanalyzing molecules. Its principle is based on detecting light scatteredoff a molecule that is shifted in energy with respect to the incidentlight. The shift, called Raman shift, is characteristic of individualmolecules, reflecting their vibrational frequencies like molecularfingerprints. As a result, the key advantage of Raman spectroscopy isits molecular specificity while its main limitation is the small signaldue to low quantum yield of Raman scattering. One way to enhance theRaman signal is to tune the excitation wavelength to be on resonancewith an electronic transition, so called resonance Raman scattering.This can usually produce an enhancement on the order of 10²-10³.

Another technique to enhance Raman scattering is surface enhancement byroughened metal surfaces, notably silver and gold, that provides anenhancement factor on the order of 10⁶-10⁸. This is termed surfaceenhanced Raman spectroscopy (SERS). Similar or somewhat largerenhancement factors (˜10⁸-10¹⁰) have been observed for metal, mostlysilver or gold, nanoparticles.

In the last few years, it has been shown that an even larger enhancement(˜10¹⁰-10¹⁵) is possible for aggregates of metal nanoparticles (MNPs),silver and gold. The largest enhancement factor of 10¹⁴-10¹⁵ has beenreported for rhodamine 6G (R6G) on single silver nanoparticleaggregates. This huge enhancement is thought to be mainly due tosignificant enhancement of the local electromagnetic field of thenanoparticle aggregate that strongly absorbs the incident excitationlight for the Raman scattering process. With such large enhancement,many important molecules that are difficult to detect with Ramannormally can now be easily detected. This opens many interesting and newopportunities for detecting and analyzing molecules using SERS withextremely high sensitivity and molecular specificity.

SERS can also be developed into a molecular imaging technique forbiomedical and other applications. Existing Raman imaging equipmentshould be usable for SERS imaging. SERS will provide a much-enhancedsignal and thereby significantly shortened data acquisition time, makingthe technique practically useful for medical or other commercial andindustrial applications including chip inspection or chemicalmonitoring. SERS is also useful for detecting other cancer biomarkersthat can interact or bind to the MNP surface. For example, Sutphen etal. have recently shown that lysophospholipids (LPL) are potentialbiomarkers of ovarian cancer (Sutphen et al., (2004) Cancer Epidemiol.Biomarker Prev. 13: 1185-1191).

For many practical applications, for example SERS and optical filters,it is highly desirable to narrow the distribution of size/shape ofnanoparticle aggregates. For SERS in particular, the incident light hasto be on resonance with the substrate absorption. Only thosenanoparticle aggregates that have resonance absorption of the incidentlight are expected to be SERS active. It is thus extremely beneficial tohave a narrow size/shape distribution and thereby narrow opticalabsorption.

Fluorescent nanoparticles (quantum dots (QDs) such as semiconductorquantum dots, SQDs) have been used recently as fluorescent biologicalmarkers and have been found to be extremely effective. They offeradvantages including higher stability, stronger fluorescence, tunabilityof color, and possibility of optical encoding based on different sizedor colored SQDs.

Metal nanoparticles have been recognized for their unique opticalproperties that could be exploited in optoelectronic devices.Nanoparticle systems composed of gold, for example, have distinctoptical properties that make them amenable to study by Raman scattering.The Raman spectrum of the adsorbed species is significantly enhanced by10 to 15 orders of magnitude when the metal nanoparticles haveaggregated, leading to enhanced electromagnetic field effects near thesurface that increases the Raman scattering intensity. The greatersensitivity found in the SERS of metal nanoparticle aggregatesfacilitates the detection and analysis of a whole host of molecules thatwere previously difficult to study.

Wang et al. disclose a method of using SQDs (dye-conjugated CdTenanoparticles, CT-NPs) to detect interactive binding between Ag-CT-NPsand Ab-CT-NPs (Wang et al., (2002) NanoLett. 2: 817-822). Theinteractions were determined by differential quenching or enhancementfluorescence activity of two different sized SQDs (red or green)measured during the analysis.

The use of SERS for analyte detection of biomolecules has beenpreviously studied. U.S. Pat. No. 6,699,724 to West et al. describes achemical sensing device and method (nanoshell-modified ELISA technique)based on the enzyme-linked immunoadsorbant assay (ELISA). The chemicalsensing device can comprise a core comprising gold sulfide and a surfacecapable of inducing surface enhanced Raman scattering (SERS). In much ofthe patent disclosure, the nanoparticle is disclosed as having a silicacore and a gold shell. The patent discloses that an enhancement of600,000-fold (6×10⁵) in the Raman signal using conjugatedmercaptoaniline was observed.

In the nanoshell-modified ELISA technique, antibodies are directly boundto the metal nanoshells. Raman spectra are taken of theantibody-nanoshell conjugates before and after the addition of a samplecontaining a possible antigen, and binding of antigen to antibody isexpected to cause a detectable shift in the spectra.

The conjugation of quantum dots to antibodies used for ultrasensitivenonisotopic detection for use in biological assays has also beenstudied. U.S. Pat. No. 6,468,808 B1 to Nie et al. disclosed an antibodyis conjugated to a water-soluble quantum dot. The binding of the quantumdot-antibody conjugate to a targeted protein will result inagglutination, which can be detected using an epi-fluorescencemicroscope. In addition, Nie et al. described a system in which aquantum dot is attached to one end of an oligonucleotide and a quenchingmoiety is attached to the other. The preferred quenching moiety in theNie patent is a nonfluorescent organic chromophore such as4-[4′-dimethylaminophenylazo]benzoic acid (DABCYL).

Raman amplifiers are also expected to be used globally as a key devicein next-generation optical communications, for example, inwavelength-division-multiplexing (WDM) transmission systems. Ramanscattering occurs when an atom absorbs a photon and another photon of adifferent energy is released. The energy difference excites the atom andcauses it to release a photon with low energy; therefore, more lightenergy is transferred to the photons in the light path.

Improving the consistency of SERS probes requires the use of single,SERS active nano-sized structures. Nano-crescents, and core-shellsystems are examples of cleverly engineered nanostructures capable ofproviding sufficient SERS intensity from individual particles due totheir ability to strongly localize surface electromagnetic fields. (Seein particular, Lu, Y., Liu, G. L., Kim, J., Mejia, Y. X., and Lee, L.P., Nano Lett. 2005, 5, 119-124; Talley, C. E., Jackson, J. B., Oubre,C., Grady, N. K., Hollars, C. W., Lane, S. M., Huser, T. R., Nordlander,P., and Halas, N. J., Nano Lett. 2005, 5, 1569-1574.) However, therelatively large size of these nanostructures will ultimately limittheir accessibility to some sub-cellular organelles. To push the sizeboundary of sensing, as required by systems biology, even smaller probeswill be required. Of interest is a subset of core-shell structures,hollow metal structures, a unique class of nanomaterials explored, mostnotably, by Sun et al. (Sun, Y. G., Mayers, B., and Xia, Y. N., AdvancedMaterials 2003, 15, 641-646). Utilizing the galvanic replacement ofsilver with gold and other metals, they have produced a variety ofdifferent sized and shaped hollow structures and have recentlydemonstrated the SERS activity of these structures (Chen, J. Y., Wiley,B., Li, Z. Y., Campbell, D., Saeki, F., Cang, H., Au, L., Lee, J., Li,X. D., and Xia, Y. N., Advanced Materials 2005, 17, 2255-2261).

In solid spherical particles there is a single resonance atapproximately 520 nm for gold and 400 nm for silver, varying slightlydepending on size and embedding media. However, when one axis isextended, for example, a nanorod, the resonance will break into twoabsorption bands, one corresponding to the short axis, or transversemode, and another to the long axis, or longitudinal mode (Nikoobakht, B.and El-Sayed, M. A., Chem. Materials 2003, 15, 1957; Chang, S. S., Shih,C. W., Chen, C. D., Lai, W. C., and Wang, C. R. C., Langmuir 1999, 15,701). The longitudinal mode has lower energy or redder absorption thanthe transfer mode. This is also true for aggregated systems in whichthere are multiple resonances within each given cluster of particles(Grant, C. D., Schwartzberg, A. M., Norman, T. J., and Zhang, J. Z., J.Am. Chem. Soc. 2003, 125, 549; Quinten, M. J., Cluster Sci. 1999, 10,319; Quinten, M., Applied Physics B-Lasers and Optics 2000, 70, 579;Quinten, M. and Kreibig, U. Applied Optics 1993, 32, 6173; Norman, T. J.Jr. Grant, C. Magma, D. Cao, D. Bridges, F. Liu, J. van Buuren, T. andZhang, J. Z., J. Phys. Chem. B 2002, 106, 7005; Norman, T. J., Grant, C.D., Schwartzberg, A. M., and Zhang, J. Z., Opt. Mat. 2005, 27, 1197; andKreibig, U. Optical properties of metal clusters; Springer: Berlin;N.Y., 1995; Vol. 25). Therefore, controlling size and shape of thesemetal nanostructures allows control of their optical properties thathave potential applications in nanophotonics and sensing.

As an effort to engineer so-called “hot spots” of large enhancement insingle particles, Lee et al. produced nano-crescent structures bydepositing silver over latex beads on a surface, then dissolving awaythe bead (Lu, Y., Liu, G. L., Kim, J., Mejia, Y. X., and Lee, L. P.,Nano Lett. 2005, 5, 119). These hollow spheres are open-ended with asharpened edge that greatly enhances the EM field. This engineered“hot-spot” approach yields improved SERS enhancements over core/shellsystems and is of a similar homogeneity due to the highly consistentlatex beads available. For applications requiring extremely small probesize, however, both nano-crescents and core shell systems are relativelylarge.

A system of particular interest where probe size is of utmost importanceis intracellular studies (Chithrani, B. D., Ghazani, A. A., and Chan, W.C. W., Nano Lett. 2006, 6, 662-668). It has been found that whileparticles larger than 100 nm can enter a cell, they do not do so readilyand may interrupt some cellular functions. Similarly, particles that aretoo small, less than 20 nm, will diffuse out of the cell, rendering themuseless. The ideal is a structure that can be tuned in size between 20nm and 100 nm depending on the application.

Nanotubes of all shapes and sizes have become an area of increasinginterest for applications ranging from filtration to electricalinterconnects. (See, in particular, Holt, J. K. et al., Science 312,1034-1037 (2006); Hinds, B. J. et al., Science 303, 62-65 (2004); Zhang,M. et al. Science 309, 1215-1219 (2005); and Huang, Y. et al., Science294, 1313-1317 (2001).) The application of these structures is almostunlimited, however, as is the case with most synthesized structures ofthis scale, nanoscopic manipulation is challenging. While carbonnanotubes have been the predominant structure of interest, lately therehas been an effort to utilize gold and silver nanotubes or nanowires forthese purposes as their conductivity and material properties are thoughtto be superior (Siwy, Z. et al., J. Am. Chem. Soc. 127, 5000-5001(2005); Kohli, P., Wharton, J. E., Braide, O. & Martin, C. R. J.,Nanosci. Nanotechnol. 4, 605-610 (2004)). Generally these metalstructures are produced by a physical or electroless depositiontechnique, and while this produces well defined structures, their shapeand size is entirely dependent on the template on which they are made,limiting the size and practical application of these structures (Wiley,B., Sun, Y. G., Mayers, B. & Xia, Y. N., Chemistry-a European Journal11, 454-463 (2005); Wiley, B. et al., M. R. S. Bull., 30, 356-361(2005); Sun, Y. G. & Xia, Y. N. Advanced Materials 16, 264-268 (2004);and Lee, M., Hong, S. C. & Kim, D., Appl. Phys. Lett., 89 (2006)).

There is therefore a need in the art for use in the chemical andbiomedical analytical industries and the electronic communicationsindustries to provide more sensitive compositions and devices that areinexpensive to manufacture and easy to use.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides nanostructures comprising hollow metalnanospheres or nanoshells and nanotubes for use as chemical sensors,conduits for fluids, and electronic conductors. The nanostructures canbe used in microfluidic devices, for detecting chemicals inside oroutside a biological membrane, such as a cell membrane or a viral coat,for transporting fluids between devices and structures in analyticaldevices, for conducting electrical currents between devices andstructure in analytical devices, and for conducting electrical currentsbetween biological molecules and electronic devices, such as microchips.

In one embodiment the present invention provides a chemical sensorcomprising gold nanoshells (hollow gold nanospheres; HGN), thenanoshells having a mean particle size of between about 20 nm and about100 nm diameter. In one preferred embodiment the mean diameter isbetween about 20 nm to about 70 nm. In another preferred embodiment, themean diameter is between about 22.8 nm and about 50 nm diameter. In oneembodiment, the invention provides a chemical sensor for chemical andbiological sensing applications, particularly those requiring near-IRabsorption.

The HGNs have an interior wall surface diameter and an exterior wallsurface diameter thereby defining the wall thickness. The inventionfurther provides HGNs having tunable interior and exterior and whereinthe peak of the surface plasmon band absorption is between about 550 nmand about 820 nm. In one embodiment the mean wall thickness of the HGNsis between about 2.4 nm and about 7.3 nm. In a preferred embodiment themean wall thickness is about 5 nm.

In another preferred embodiment, the chemical sensor has a surfacewherein the surface can induce surface enhanced Raman scattering (SERS).

In still another preferred embodiment, the chemical sensor furthercomprises at least one detecting molecule, wherein the detectingmolecule is bound to the surface. In a more preferred embodiment thedetecting molecule is selected from the group consisting of proteins,peptides, antibodies, antigens, nucleic acids, peptide nucleic acids,sugars, lipids, glycophosphoinositols, and lipopolysaccharides.

In a yet more preferred embodiment the detecting molecule is anantibody. In another preferred embodiment, the detecting molecule is anantigen.

In another embodiment, the invention provides a chemical sensor furthercomprising at least one semiconductor quantum dot. In a preferredembodiment the semiconductor quantum dot further comprises a linkermolecule, the linker molecule selected from the group consisting of athiol group, a sulfide group, a phosphate group, a sulfate group, acyano group, a piperidine group, an Fmoc group, and a Boc group.

In a still further embodiment, the invention provides a chemical sensorcomprising at least one semiconductor quantum dot wherein thesemiconductor quantum dot further comprises a detecting molecule,wherein the detecting molecule is bound to the semiconductor quantumdot. In a more preferred embodiment, the detecting molecule is selectedfrom the group consisting of proteins, peptides, antibodies, antigens,nucleic acids, peptide nucleic acids, sugars, lipids,glycophosphoinositols, and lipopolysaccharides.

In a more preferred embodiment, the detecting molecule is an antibody.In the alternative, a more preferred embodiment comprises a chemicalsensing device wherein the detecting molecule is an antigen.

Another embodiment of the invention provides a method for detecting ananalyte in a sample using a chemical sensor, the method comprising thesteps of: i) providing a sample; ii) providing a semiconductor quantumdot comprising a linker molecule (LM-SQD); iii) conjugating the analytein the sample with the LM-SQD thereby producing an analyte-LM-SQDconjugate; iv) providing a chemical sensor comprising a plurality ofparticles, each particle comprising: a shell having at least one surfaceand wherein the shell comprises a gold molecular species, the shellsurface further comprising a detecting molecule; v) incubating theanalyte-LM-SQD conjugate with the chemical sensor for a predeterminedtime period; and vi) measuring the extent of binding between theanalyte-LM-SQD conjugate and the chemical sensor; thereby detecting theanalyte in the sample. In one embodiment the sample is selected from thegroup consisting of mammalian cells, vertebrate cells, invertebratecells, plant cells, fungal cells, mold cells, archaeal cells, bacterialcells, viruses, bacteriophages, and the like. In another embodiment thesample is selected from the group consisting of blood fluids, lymphfluids, hemolymph fluids, pulmonary surfactant fluids, peritonealfluids, gastric fluids, xylem fluids, phloem fluids, and the like. Inyet another embodiment the sample is selected from the group consistingof fluvial fluids, marine fluids, atmospheric precipitate fluids,waste-water fluids, agricultural run-off fluids, fluids comprisinghydrocarbons, fluids contaminated by hydrocarbons, aerosol fluids,aqueous fluids, non-aqueous fluids, and the like.

The invention also provides a method of using the chemical sensor asdisclosed herein for measuring cellular processes. These embodiments aremerely exemplary of the invention, which encompasses any smallnanostructures having SERS activity as disclosed herein.

In another embodiment the invention provides a method for detecting ananalyte that is a cancer marker. In one embodiment the cancer marker isan antibody. In one embodiment of the invention the detecting moleculein the chemical sensor is an antigen that binds to a cancer markerantibody with an affinity (K_(a)) of at least 10⁶ l/mole. In a morepreferred embodiment the K_(a) is at least 10⁸ l/mole. In anotherpreferred embodiment the analyte is a phospholipid. In a most preferredembodiment the phospholipid is lysophosphatidic acid (LPA).

The invention further provides a synthetic nanotube, the syntheticnanotube being substantially hollow and having dimensions of betweenabout 20 nm and about 100 nm in mean diameter and at least between about0.1 μm and 4 μm in mean length. In a more preferred embodiment the meandiameter is between about 30 nm and 80 nm. In a more preferredembodiment the mean length is between about 4 μm and about 50 μm, forexample, about 6 μm, about 8 μm, about 10 μm, about 15 μm, about 20 μm,about 25 μm, about 30 μm, about 40 μm, and about 50 μm, and any otherlength therebetween.

In one embodiment the synthetic nanotube has a wall of mean dimension ofbetween about 2.4 nm and about 7.3 nm across. In a preferred embodimentthe wall has a mean dimension of about 5 nm.

In one preferred embodiment the synthetic nanotube comprises a metalselected from the group consisting of gold, silver, platinum, copper,aluminum, palladium, cadmium, iridium, rhodium, and the like.

In another embodiment, the invention provides a conduit for conductingfluids, the conduit comprising the synthetic nanotube as disclosedherein.

In yet another embodiment, the invention provides an electronicconductor, the electronic conductor comprising the synthetic nanotube asdisclosed herein.

The invention further provides a method for synthesizing a nanotube, thenanotube comprising a metal, the method comprising the steps of (i)combining an aqueous solution of Co²⁺ salt with an aqueous solution ofcitrate salt thereby forming a first mixture (ii) degassing the firstmixture; (iii) purging at least once with nitrogen gas; (iv) adding anaqueous solution of NaBH₄ thereby reducing the Co²⁺ to Co⁰, and therebyforming a second mixture comprising Co⁰ particles, the step of addingbeing in the presence of an induced magnetic field and wherein thepresence of the induced magnetic filed aligns the Co⁰ particles; (v)agitating the second mixture until hydrogen evolution is substantiallycomplete; (vi) adding the second mixture comprising aligned Co⁰particles to an aqueous solution of Au³⁺ salt; (vii) allowing the Au³⁺to be reduced to Au⁰ and the Co⁰ oxidized to Co²⁺, and wherein the Au⁰is deposited adjacent to the aligned Co⁰ thereby creating a nanotubecomprising Au⁰, the method thereby synthesizing a nanotube. In onepreferred embodiment the nanotube comprises a metal selected from thegroup consisting of gold, silver, platinum, copper, aluminum, palladium,cadmium, iridium, and rhodium.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1 shows an exemplary synthesis procedure for HGNs.

FIG. 2 illustrates the different tunable colors of the HGNs havingcombinations of different dimensions.

FIG. 3 illustrates the UV-visible electronic absorption spectra ofdifferent HGNs.

FIG. 4 shows a comparison of SERS spectrum consistency between HGNs andsilver nanoparticles/aggregates. Shown are the single particle SERSspectrum of MBA on HGNs (red trace, top) and silver aggregates (bluetrace, bottom). The inset is a histogram of the relative intensity ofthe two most intense peaks of MBA at 1070 cm⁻¹ and 1590 cm⁻¹ of 150 HGNs(red bars) and 150 silver aggregates (blue bars).

FIG. 5 illustrates a correlation between confocal SERS (a) and TEM (b).

FIG. 6 is a representative low resolution TEM of HGNs. Examining 150particles from such images, the mean size is found to be 30±4.5 nm.

FIG. 7 is a high resolution TEM of an individual HGN of diameter 29.1 nmwith approximately 5 nm wall thickness. Twinning in the HGN walldemonstrates its polycrystalline nature. A TEM of the whole HGNs isinset.

FIG. 8 illustrates the ensemble average solution absorption spectrum ofan as prepared solution (black trace, top), and the Rayleigh scatteringspectrum of a single HGNs (red trace, middle) immobilized on a glasscoverslip in air. Plotted against the right axis is a histogram of thepeak wavelength in the scattering spectra (l_(max)) of 100 particles(average 621±10.6 nm) (blue bars, bottom). The absorption spectrum isshifted in intensity for clarity. Inset: Rayleigh scattering spectra oftwo silver aggregates.

FIG. 9 is a TEM image of individual HGNs on a holey carbon TEM grid ofwhich, the SERS spectrum has been measured (inset). TEM image wasoverlapped with confocal Raman images to co-locate the SERS activeparticles shown. Light region of the image is a hole in the film. Redcircle marked “Focal Area” represents the approximate diameter of thelaser focal area used to measure the SERS response of the sample.

FIG. 10 represents each black point as the intensity of the pH sensitive1430 cm⁻¹ peak of 20-30 particles at different pH normalized to the pHinsensitive 1590 cm⁻¹ peak, while the red crosses are the individualdata points. Error bars represent standard deviation of themeasurements. The fit line is a guide to the eye.

FIG. 11 illustrates particle size as a function of citrate and sodiumborohydride concentration. All particle sizes are determined byexamining TEM images of the resulting gold structures and represent themeasurement of at least 200 particles. Reported sizes are the particlediameter.

FIG. 12 illustrates histograms showing the size dispersion of cobaltnanoparticles produced by slow and fast addition of cobalt chloride.Solid lines are best fits demonstrating particle dispersion. Particlessizes determined by measuring low resolution TEM images.

FIG. 13 show transmission electron micrographs of the HGNs. FIG. 13A isa high resolution TEM of a single, 30 nm HGN. The wall thickness isapproximately 4 nm and large areas of crystallinity are clearly visible.FIGS. 13B-E are low resolution TEM images of particles of 71±17 nm (B),50±5 nm (C), 40±3.5 nm (D), and 28±2.3 nm.

FIG. 14 illustrates the UV-visible absorption spectra of nine HGNsamples with varying shell diameters and wall thicknesses.

FIG. 15 shows plasmon absorbance maximum wavelength (λ_(max)) as afunction of shell thickness and shell diameter. Each point represents anindividual set of experiments and represents the average measuredlengths.

FIG. 16 illustrates spectral dependence on volume of added gold salt.Gold solutions were diluted to 10 ml with water before 30 ml of a cobaltsolution made by the fast cobalt addition method with 100 μl sodiumborohydride and 600 μl of citric acid. Average particle size is 35±2 nm.

FIG. 17 illustrates a comparison of ensemble averaged absorption andsingle particle Rayleigh scattering of 30±2.6 nm HGNs.

FIG. 18 illustrates the ensemble averaged surface enhanced Ramanscattering spectrum of mercaptobenzoic acid on the HGNs.

FIG. 19 is a high resolution TEM of an HGN formed from a slightlyoxidized cobalt particle.

FIG. 20 illustrates TEMs of the gold nanotubes. FIG. 20 a is a lowresolution TEM image of gold nanotubes. Red line indicates the path of asingle ˜4 mm tube. FIG. 20 b is a high resolution TEM image of a largesection of one tube illustrating the continuity and consistency of thesamples. FIG. 20 c is a high resolution TEM image of one section of thegold tube showing its continuous nature. FIG. 20 d is a more detailedhigh resolution TEM image of the tube showing gold lattice fringesindicating its poly-crystalline nature.

FIG. 21 is a schematic of gold tube formation templated with Conanoparticles aligned by an external magnetic field. Top portion offigure: alignment of cobalt nanoparticles along magnetic field lines.Middle portion: Gold is reduced onto the surface of aligned cobaltparticles. Bottom portion: Cobalt is further oxidized by dissolvedoxygen leaving a hollow structure.

FIG. 22 illustrates the UV-visible absorption spectra of 50 nm diametersolid gold nanoparticle solution, 40 nm diameter nanotube with ˜5 nmshell thickness solution, and 60 nm diameter HGN with 3 nm shellthickness solution.

FIG. 23 illustrates how the terminal amino acid residues in differentpeptides affect the SERS spectrum. The SERS spectra change when theterminal amino acid residue is changed thereby indicating a possiblerelationship between SERS spectrum and distance between the terminalresidues. Y=tyrosine; P=proline, and W=tryptophan.

FIG. 24 illustrates cross-sectional representations different hollownanosphere or nanoshell structures. The same structures can beconsidered for nanotubes in cross-section.

DETAILED DESCRIPTION OF THE INVENTION

The embodiments disclosed in this document are illustrative andexemplary and are not meant to limit the invention. Other embodimentscan be utilized and structural changes can be made without departingfrom the scope of the claims of the present invention.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, a reference to “a particle” includes aplurality of such particles, and a reference to “a surface” is areference to one or more surfaces and equivalents thereof, and so forth.

The invention disclosed herein is based on the galvanic replacement ofcobalt with gold, a procedure shown to produce considerably morehomogeneous hollow spheres than those synthesized with silver (Liang, H.P.; Wan, L. J.; Bai, C. L.; Jiang, L. J., Phys. Chem. B 2005, 109,7795-7800). The process starts with a cobalt nanoparticle that issynthesized as a template for the growth of hollow gold nanospheres(HGNs). Utilizing the difference in redox potential between cobalt andgold, it is possible to reduce gold ions while oxidizing the cobaltnanoparticles. Because this reaction takes place entirely at the surfaceof the cobalt particle, the shape and size of the resulting hollowstructure is completely dependent on the original template. Moreover,this process is able to produce SERS active nanoparticles that aresignificantly smaller than traditional nanoparticle structures used forSERS, providing a sensor element that can be more easily incorporatedinto cells for localized intracellular measurements.

We provide the successful SERS application of HGNs with improved opticaland structural homogeneity over other SERS substrates that are highlydesired and important for size sensitive biological sensingapplications. The consistency of particle shape and size is reflected inthe optical properties that lead to a tenfold increase in SERS spectralconsistency over aggregated silver nanoparticles commonly used in SERSapplications. SERS from single HGNs was achieved, the first suchmeasurement on hollow nanostructures. Finally, pH sensing as a modelsystem was demonstrated showing an approximate doubling of resolutionand a ten-fold increase in precision over previous nano-sized pH SERSprobes. This clearly represents a new detecting platform and a majorstep forward in potential biological sensing applications.

Since the early work by Turkevich et al. and later Frens et al., it hasbeen understood that in a standard colloidal gold synthesis using thehot citrate reduction of chloroauric acid, the particle size may becontrolled by the concentration of citrate. Citrate stabilizes theinitially formed nuclei and the more citrate is present, the more nucleiwill be stabilized. However, when trying to apply this logic to theaqueous synthesis of cobalt nanoparticles, it is a significantly morechallenging task. (See Turkevich, J.; Stevenson, P. C.; Hiller, J.,Discussions of the Faraday Society 1951, 11, 55; Frens, G., NaturePhysical Science 1973, 241, 20.)

Due to the stability of the cobalt salt, the reduction cannot be done bycitrate alone and a stronger reducing agent is required. In this casesodium borohydride is used to reduce the salt, and citrate is presentonly as a capping agent.

Nearly monodisperse HGNs with tunable interior and exterior diameterhave been synthesized by sacrificial galvanic replacement of cobaltnanoparticles. By carefully controlling particle size and wallthickness, it is possible to tune the peak of the surface plasmon bandabsorption between 550 nm and 820 nm. Cobalt particle size is tunable bysimultaneously changing the concentration of sodium borohydride andsodium citrate, the reducing and capping agent, respectively. Thethickness of the gold shell can be varied by carefully controlling theaddition of gold salt. With successful demonstration of ensemble as wellas single HGN surface enhanced Raman scattering, these HGNs have showngreat potential for chemical and biological sensing applications,especially those requiring nanostructures with near IR absorption.

In this application we present the synthetic route necessary to controlthe particle size of the cobalt nanoparticles, which is reflected in theresultant HGN diameter. The inner diameter, or wall thickness, can becontrolled by the concentration of gold salt used, leading to completecontrol of the optical properties of particles ranging from 20 nm to 70nm. For a particular diameter and wall thickness, the absorption band isrelatively narrow due to the near monodisperse distribution, asdetermined by single nanosphere scattering spectrum. These HGNs havebeen further demonstrated to be excellent SERS substrates with excellentconsistency measured based on single HGN SERS spectrum.

The size of the particles can be in the range from between about 20 nmto about 100 nm, about 25 nm to about 85 nm, about 35 nm to about 70 nm,and about 50 nm in diameter. The dimensions of the wall of the particle,that is the wall thickness, is in the range form about 2.4 nm to about10 nm, from about 2.4 nm to about 7.3 nm, and about 5 nm thick.

An exemplary method for synthesizing HGNs is illustrated on FIG. 1. FIG.2 illustrates the different color spectra associated with HGNs ofdifferent combinations of shell thickness and outer shell diameter. FIG.3 exemplifies the UV-visible electronic absorption spectra of HGNshaving a variety of combinations of shell thickness and outer diameterFIG. 5 compares a confocal image (labeled a) with that of a TEM (labeledb) of a sample of nanospheres. The inset photomicrographs show a highresolution image of Particle A and of Particle B; the graphs below showthe SERS spectrum associated with Particle A or Particle B which can bediscretely distinguished. As used herein, the terms “nanoshells” and“hollow nanospheres” are interchangeable.

SERS Detection Applications for Sensing and Imaging

Raman spectroscopy is a powerful optical technique for detecting andanalyzing molecules. Its principle is based on detecting light scatteredoff a molecule that is shifted in energy with respect to the incidentlight. The shift, called Raman shift, is characteristic of individualmolecules, reflecting their vibrational frequencies that are like figureprints of molecules. As a result, the key advantage of Ramanspectroscopy is its molecular specificity while its main limitation isthe small signal due to low quantum yield of Raman scattering. One wayto enhance the Raman signal is to tune the excitation wavelength to beon resonance with an electronic transition, so called resonance Ramanscattering. This can usually produce an enhancement on the order of10²-10³. Another technique to enhance Raman scattering is surfaceenhancement by roughened metal surfaces, notably silver and gold, thatprovides an enhancement factor on the order of 10⁶-10⁸. Similar orsomewhat larger enhancement factors (˜10⁸-10¹⁰) have been observed formetal, mostly silver, nanoparticles.

In the last few years, it has been shown that an even larger enhancement(˜10¹⁰-10¹⁵) is possible for aggregates of metal nanoparticles, forexample, comprising silver and/or gold. The largest enhancement factorof 10¹⁴-10¹⁵ has been reported for rhodamine 6G (R6G) on single silvernanoparticle aggregates. This huge enhancement is thought to be mainlydue to significant enhancement of the local electromagnetic fields ofthe nanoparticle aggregates that absorb strongly the incident excitationlight for the Raman scattering process. With such large enhancement,many important molecules that are difficult to detect with Ramannormally can now be easily detected. This provides many interesting andnew opportunities for detecting and analyzing molecules using SERS withextremely high sensitivity and molecular specificity.

SERS can also be developed into a molecular imaging technique forbiomedical and other applications. Exciting Raman imaging equipment maybe usable for SERS imaging. SERS can provide an enhanced signal andthereby significantly shortened data acquisition time, making thetechnique practically useful for medical or other commercial andindustrial applications including, but not limited to, chip inspectionor chemical monitoring.

Antigen/Antibody Detection with Metal and Semiconducting Nanoparticles

Fluorescent nanoparticles (semiconductor quantum dots, SQDs) have beenused recently as fluorescent biological markers and have been found tobe extremely effective. They offer advantages including higherstability, stronger fluorescence, tunability of color, and possibilityof optical encoding based on different sized or colored SQDs.

HGNs of the invention can be used to detect an analyte. Such an analytecan be, for example, but not limited to, an antigen, an antibody, abiochemical metabolite, an organic compound, a compound or elementhaving biological activity, or the like.

SERS is also useful for detecting other cancer biomarkers that caninteract or bind to the HGN surface. For example, Sutphen et al. haverecently shown that lysophospholipids (LPL) are potential biomarkers ofovarian cancer (Sutphen et al., (2004) Cancer Epidemiol. BiomarkerPrev., 13: 1185-1191). Based on the molecular structure of LPLmolecules, a favorable interaction between LPL molecules with HGNthrough electrostatic interaction can occur at the appropriate pH. Inthe case of the SERS experiment using a polyclonal Ab, the strongestinteraction with HGN occurs at the isoelectrostatic pH, i.e. pH at whichthe HGN has equal number of positive and negative charges. The pH isvaried to adjust the charge on the HGN to determine the optimal pH orcharge for strong interaction with LPL.

By conjugating fluorescent nanoparticle QDs to antigens and mixing theAg-QD conjugate with a HGN-Ab composition, quenching of fluorescenceupon binding of the antigen/antibody pair can be observed. The Ag and/orthe Ab can be conjugated to the QD or HGN using a linker molecule (LM).A decrease in fluorescence can indicate the presence of the antibody forthat particular antigen to which the fluorescing QDs have been attached.Depending on which antigen is utilized a wide array of antibodies can bedetected. This can allow for the rapid detection of cancers or diseasesthat currently can take days or weeks to diagnose. Likewise, the schemecan work as well if antibody is attached to a fluorescent QD and therespective antigen to a metal nanoparticle. Metal particles have noflorescence with visible excitation. The fluorescence quenching by metalnanoparticles can be more effective than quenching by larger QDs. Thisapproach is sensitive and specific. The distance between the metalnanoparticle and QD is important for this to work (for example, thedistance can be less than 2 nm). The interaction between the twocomponents can be adjusted to achieve the maximum quenching effect.

Detection of Tumor Markers

Surface-enhanced Raman scattering using silver nanoparticles was appliedto detect various forms of lysophosphatidic acid (LPA) to examine itspotential application as an alternative to current detection methods ofLPA as biomarkers of ovarian cancer. Enhancement of the Raman modes ofthe molecule, especially those related to the acyl chain within the800-1300 cm⁻¹ region, was observed. In particular, the C—C vibrationmode of the gauche-bonded chain around 1100 cm⁻¹ was enhanced to allowthe discrimination of two similar LPA molecules. Given the molecularselectivity of this technique, the detection of LPA using SERS mayeliminate the need for partial purification of samples prior to analysisin cancer screening.

Lysophosphatidic acid (LPA), originally known for its role as anintermediate in intracellular lipid metabolism, has now been recognizedas an important multifunctional biological mediator that can elicitcellular responses including mitogenic and antimitogenic effects on thecell cycle, actin skeleton regulation, and cellular motility (see Tigyiet al., (1994) Proc. Nat. Acad. Sci. 91: 1908-1912; van Corven et al.,(1989) Cell 59: 45-54; Ridley and Hall, (1992) Cell 70: 389-399; andZhou et al., (1995) J. Biol. Chem. 270: 25549-25556). The involvement ofLPA in inducing cell proliferation, migration and survival implicates itin the initiation and progression of malignant disease, and has beenproposed as a sensitive biomarker for ovarian cancer (see Xu et al.(1998) JAMA 280: 719-723; Mills and Moolenaar (2003) Nature Rev. 3:582-591; Fang et al. (2004) J. Biol. Chem. 279: 9653-9661; and Sutphenet al. (2004) Cancer Epidemiol. Biomark. Prev. 13: 1185-1191).

Typically, the detection of LPA has been conducted using chromatographyand mass spectroscopy assays that require a partial purification of thesamples using thin layer chromatography (TLC) prior to analysis.Although this method is effective, an underestimation of LPAconcentration can result during the recovery process due in part to thevarying mobility of the LPA salts (free acid, sodium and calcium salts)when subjected to chromatography by TLC. The low stability of LPA alsocalls for fast and sensitive detection techniques.

A powerful optical detection technique based on surface-enhanced Ramanscattering (SERS) offers a unique combination of high sensitivity andmolecular specificity. With SERS, the Raman signal of a molecule isincreased by many orders of magnitude as a result of strong enhancementof the excitation light through the resonance of the metal's surfaceelectrons called the surface plasmon (see Moskovitz (1985) Rev. ModernPhysics 57: 783-828; Otto et al., (1992) J. Phys. Condense Matter 4:1143-1212; and Campion and Kambhampati, (1998) Chem. Soc. Rev. 27:241-250). SERS has been successfully used in the detection and analysisof a large number of chemicals and biological molecules (see Albrechtand Creighton, (1977) J. Am. Chem. Soc. 99: 5215-5217; Nie and Emory(1997) Science 275: 1102-1106; Keating et al., (1998) J. Phys. Chem. B102: 9414-9425; Kneipp et al., (1998) Phys. Rev. E 57: R6281-R6284; andSchwartzberg et al., (2004) J. Phys. Chem. B 108: 19191-19197).

SERS Application for Detection and Analysis of SemiconductorNanoparticles

Another application of SERS based on the gold nanoparticle system is formeasuring Raman spectrum of semiconductor nanoparticles (QDs). Similarto molecules, normal Raman signals are very small and thus Ramanspectrum is challenging to measure. SERS as an enhanced Raman techniquefor measuring Raman for semiconductor nanoparticles have not beenreported before. The surface chemistry of the metal nanoparticles andthe semiconductor QDs must be compatible for this to work. The sulfurspecies on the surface of the HGNs are ideal for II-VI SQDs to bind,enabling SERS detection of the SQDs. This provides a powerful method fordetecting and analyzing semiconductor nanoparticles.

The material and methods described heretofore have additional propertiesand uses. As such, we herein disclose an aqueous solution phasesynthesis of continuous gold nanotubes that are controllable in shapeand size, currently up to 5 mm in length, by magnetic field manipulationand synthetic parameters. Because of the ease with which magnetic fieldsmay be manipulated, precise placement should not only be possible, butrelatively simple as compared to other methods. This is the first stepin producing controllable, well-defined, chemically stable structuresfor any application that requires hollow, electrically conductingone-dimensional nanomaterials.

Use of Nanostructures in Miniature Electrical Circuits

The nanostructures in the form of nanotubes can be used in theproduction of miniature electronic circuits, with applications in themicroelectronics industries for producing very small circuits for memorychips, for creating electrical circuits used with biological media, suchas proteins, including cell surface receptor proteins, antibodies;photosensitive compounds, such as chlorophyll and related compounds;xanthocyanins; compounds having oxidoreductase activity, including, butnot limited to, cytochromes and related compounds, haemoglobin,myoglobin, and the like, and fluorescent compounds.

Synthesis of Nanotubes using Magnetic Alignment of Metal Particles

In previous works we and others have shown that it is possible toproduce highly uniform hollow gold nanospheres (HGN) using synthesizedcobalt nanoparticles in aqueous solution as sacrificial electrolessdeposition templates (Schwartzberg, A. M., Olson, T. Y., Talley, C. E.and Zhang, J. Z. J. Phys. Chem. B 110, 19935-19944 (2006)). In thisreaction Au³⁺ is reduced to Au⁰ by Co⁰ nanoparticles via the followingmechanism:

3Co⁰±2Au³⁺→3Co²⁺+2Au⁰

Because only two gold atoms are generated for every three cobalt atomsoxidized, there is a net loss of volume, resulting in goldnanostructures of approximately ⅔ the volume of the original Conanoparticle. Since the gold shell grows inward from the surface of thecobalt particle, a void remains at the center of the final goldnanostructure that is filled, most likely, by water and various ionicspecies. The shape and size of these hollow structures therefore dependsstrongly on the original cobalt nanostructure.

We have found that the size and shell thickness of HGNs can be rathersimply controlled by synthetic methods (Schwatzberg (2006) supra). Inattempts to increase particle size, and thereby red-shift the resultingHGN plasmon into the near IR, it was found that reducing theconcentration of sodium citrate, the particle stabilizing agent, andincreasing the concentration of the cobalt salt results in acontrollable aggregation of the cobalt nanoparticles. By carefullyvarying the citrate concentration we were able to induce varying statesof aggregation from complete flocculation to partial crosslinking.Surprisingly, in the presence of a relatively strong magnetic field, amagnetic stir-plate or the like, upon reaction with the gold salt, theseweakly aggregated cobalt nanoparticles were found to produce long, wellorganized gold tubes as in FIG. 20 a. While the alignment of cobaltnanoparticles has been observed in the past, this is the first time thephenomenon has been used to create extended gold nanostructures (Puntes,V. F., Krishnan, K. M. and Alivisatos, A. P., Science 291, 2115-2117(2001); Salgueirino-Maceira, V. and Correa-Duarte, M. A., J. Mat. Chem.16, 3593-3597 (2006); and Salgueirino-Maceira, V., Correa-Duarte, M. A.,Hucht, A. and Farle, M., J. Magnetism Magnet. Mat. 303, 163-166 (2006)).

Such a surprising occurrence would not have been predicted and isclearly an example of superior unexpected results when compared withwhat is known in the prior art.

The highlighted tube in FIG. 20 a is over 4 μm long and upon closeinspection, the location of each individual cobalt particle thatoxidized to form the gold tube is clear. This leads to the almost peapodor intestine-like structure of the tube and it is clear that the cobaltparticles are aligning into a “string of pearls” type structure. Fromlow resolution TEM images it is not entirely clear if each section ofthe “intestine” is divided by walls, or if the hollow portion of thetube is continuous. High resolution TEM, however, conclusively showsthat in most cases the whole tube is hollow and polycrystalline innature, that is, nanotubes, not rods or wires (see FIGS. 20 b, c, andd). We thought initially that these may be aggregates of goldnanoshells, which would be easier to explain. However, closerexamination by HRTEM shows that this is not the case since the hollowpart of the structure is connected throughout the whole tube (FIGS. 20 band c) and the lattice fringes of each “section” extend into the nextindicating simultaneous growth (FIG. 20 d). Therefore, the nanotubestructure is clearly not simply aggregates of gold nanoshells.Furthermore, the apparent one-dimensional (or linear) structure isinconsistent with randomly aggregated structures that tend to bethree-dimensional. The linear structure suggests that something isdirecting the formation of the nanotube structure in an ordered manner.

One possible clue to explaining the formation of the Au nanotubes comesfrom the magnetic property of the seeding Co nanoparticles and theapplication of an external magnetic field to drive a magnetic stir barduring synthesis. To test this, we then intentionally examined theeffect of an external magnetic field on the synthesis process and thefinal product. We have found that the magnetic field has a stronginfluence on the formation of the nanotubes. Without an externalmagnetic field we observed little or no nanotube formation. This led usto suggest that the mechanism of growth depends on the ordering of theCo nanoparticles by a magnetic field. The initial finding of nanotubeswas the result of an unintended use of a magnetic stir bar and platethat uses a strong magnetic field that caused alignment of the Conanoparticles. Even though Co nanoparticles are magnetic and could alignby themselves, this self-alignment is apparently insignificant at roomtemperature. When the magnetic field is strong enough, the Conanoparticles align into chains along the field lines by the appliedexternal magnetic field, as illustrated in FIG. 21. Reduction of Au saltinto Au metal with these aligned Co chains resulted in the formation ofthe Au nanotube. The Co nanoparticles are close enough that the Au saltcannot access the interstitial space between the particles resulting ina structure with a completely hollow core.

It is possible not only to control the cobalt particle before reactingwith gold. By performing the reaction in an oxygen free environment andusing only enough gold to oxidize part of the cobalt particles, we wereable to leave some portion of the magnetic particles at the core of thegold tube. The tube is then magnetically controllable and can be alignedin whatever way desired by magnetic field manipulation. By merelyexposing the tubes to oxygen, the cobalt was completely oxidized withinseconds and dissolved into solution, leaving a completely hollow tube.

Given the nature of the tube synthesis there are always pores that allowgold ions to permeate in to react with the cobalt and to allow oxidizedcobalt to diffuse out. It is important to note that for applicationsrequiring pore free tubes, it is simple to backfill the holes after theoxidation of cobalt is complete. By adding additional gold salt in thepresence of sodium citrate, a seed mediated growth mechanism will inducereduction of gold at the tube surface, specifically at sharp featuressuch as pores. With this process it is possible to form smooth surfacedtubes and, with sufficient gold salt, grow the wall thicker if desired.The potential applications of this type of controllable, conductingnanostructure are numerous.

In addition, we have found that the average length of the tubes isdependent on the amount of initial cobalt aggregation chemicallyinduced. By decreasing the amount of sodium citrate it is possible tolengthen the tubes. With this, we have additional control of thestructures. Length and diameter can be controlled chemically, wallthickness can be controlled by the amount of gold added, and order andplacement can be controlled magnetically. It should be noted that tubesare not formed when sufficient citrate is present to preventaggregation. This indicates that, while the particles may align in thepresence of the magnetic field, they are not close enough to prevent theAu salt from penetrating the junction of the particles to reduce attheir surfaces. The synthesis has been reproduced by differentresearchers in our lab independently with very similar results. Theelectromagnetic field can be generated, for example, using a magneticstirbar and a rotating magnet system well-known to those of skill in theart. In addition, an electromagnetic field can be induced using agenerator or the like that induces an electromagnetic filed in thevicinity of the experimental particles to be aligned. Other physicalmeans for inducing an electromagnetic field are well-known to those ofskill in the art.

Associated with the novel Au nanotubular structure are some interestingoptical properties. Generally, gold nanostructures with asymmetric axes,such as nanorods, will exhibit multiple plasmon absorption bands. A redshifted longitudinal band and a transverse band to the blue associatedwith the long axis and the short axis of the structure, respectively. Athird mode perpendicular to the wall of the tube is likely too blue tobe observed due to the thinness of the wall. In the absorption spectraof these structures, however, there is only one band despite thepresence of two possible axes of plasmon oscillation (FIG. 22). Thereare two possible explanations for this. First, because of their length,the longitudinal mode is red shifted far into the near IR and is notvisible in the range in which we are looking. More likely, however,there is no surface plasmon absorption observed on the long axis becauseit is on the micron length scale and any electron oscillation is nolonger surface confined and is more bulk-like. Therefore, the absorptionpeak present in FIG. 22 must be due to oscillations around thecircumference of the tube only. This is further reinforced by thespectral position of the nanotube band between the thinly shelled 60 nmHGNs and solid gold nanoparticles. With an average diameter of 40 nm andwall thickness of ˜5 nm the circumference of the tube should yield aplasmon absorption red shifted from the HGN presented.

The mean length of the nanotube can be, for example, between about 0.1μm and about 50 μm, such as 0.1 μm, 0.2 μm, 0.5 μm, 0.75 μm, 1.0 μm, 1.5μm, 2.0 μm, 3.0 μm, 4.0 μm, 5.0 μm, 7.5 μm, 10 μm, 15 μm, 20 μm, 25 μm,30 μm, 35 μm, 40 μm, 45 μm, 50 μm, or any length therebetween. Thelength can be measured using electron microscopy and standard metricswell known to those of skill in the art. The mean wall thickness can be,for example, 0.5 nm. 1.0 nm, 1.5 nm, 2.0 nm, 2.4 nm, 2.6 nm, 3.0 nm, 4nm, 5 nm, 6 nm, 7 nm, 7.3 nm, 8 nm, 9 nm, and 10 nm, or any thicknesstherebetween The mean diameter of the nanotube can be, for example, 10nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, and 100 nm,or any diameter therebetween. The mean diameter can be measured from theimage on an electronmicrograph. The mean diameter can be measured over aportion of the entire nanotube.

In summary, we herein present a new method for producing chemicallystable and electrically conducting nanotubes. The length, diameter, andwall thickness of the nanotubes can be controlled chemically while theirposition and structural alignment can be controlled magnetically. Thismethod affords the possibility of fabricating a variety of easilymanipulated, useful linear nanotubular structures for differentapplications.

Synthesis of Biological Molecules Chemical Synthesis of Peptides

Proteins or portions thereof may be produced not only by recombinantmethods, but also by using chemical methods well known in the art. Solidphase peptide synthesis may be carried out in a batchwise or continuousflow process which sequentially adds α-amino- and side chain-protectedamino acid residues to an insoluble polymeric support via a linkermolecule. A linker molecule such as methylamine-derivatized polyethyleneglycol is attached to poly(styrene-co-divinylbenzene) to form thesupport resin. The amino acid residues are N-α-protected by acid labileBoc (t-butyloxycarbonyl) or base-labile Fmoc(9-fluorenylmethoxycarbonyl). The carboxyl group of the protected aminoacid is coupled to the amine of the linker group to anchor the residueto the solid phase support resin.

Trifluoroacetic acid or piperidine are used to remove the protectinggroup in the case of Boc or Fmoc, respectively. Each additional aminoacid is added to the anchored residue using a coupling agent orpre-activated amino acid derivative, and the resin is washed. Thefull-length peptide is synthesized by sequential deprotection, couplingof derivatized amino acids, and washing with dichloromethane and/orN,N-dimethylformamide. The peptide is cleaved between the peptidecarboxy terminus and the linker group to yield a peptide acid or amide.These processes are described in the Novabiochem 1997/98 Catalog andPeptide Synthesis Handbook (San Diego Calif. pp. S1-S20). Automatedsynthesis may also be carried out on machines such as the ABI 431Apeptide synthesizer (ABI). A protein or portion thereof may be purifiedby preparative high performance liquid chromatography and itscomposition confirmed by amino acid analysis or by sequencing (Creighton(1984) Proteins, Structures and Molecular Properties, WH Freeman, NewYork N.Y.).

In particular, a purified antigen may be used to produce antibodies orto screen libraries of pharmaceutical agents to identify those thatspecifically bind an antigen. Antibodies to an antigen may also begenerated using methods that are well known in the art. Such antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric,and single chain antibodies, Fab fragments, and fragments produced by aFab expression library. Neutralizing antibodies (i.e., those whichinhibit dimer formation) are especially preferred for therapeutic use.

For the production of polyclonal antibodies, various hosts includinggoats, rabbits, rats, mice, humans, and others may be immunized byinjection with an antigen or with any fragment or oligopeptide thereofthat has immunogenic properties. Rats and mice are preferred hosts fordownstream applications involving monoclonal antibody production.Depending on the host species, various adjuvants may be used to increaseimmunological response. Such adjuvants include, but are not limited to,Freund's, mineral gels such as aluminum hydroxide, and surface-activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemacyanin (KLH), and dinitrophenol. Amongadjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable. (For review of methodsfor antibody production and analysis, see, for example, Harlow and Lane(1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.)

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to an antigen have an amino acid sequence consistingof at least about 5 amino acids, and, more preferably, of at least about14 amino acids. It is also preferable that these oligopeptides,peptides, or fragments are identical to a portion of the amino acidsequence of the natural protein and contain the entire amino acidsequence of a small, naturally occurring molecule. Short stretches ofantigen amino acids may be fused with those of another protein, such asKLH, and antibodies to the chimeric molecule may be produced.

Antibodies

Monoclonal antibodies to an antigen may be prepared using any techniquethat provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique. (See, for example, Kohler et al. (1975) Nature256: 495-497; Kozbor et al. (1985) J. Immunol. Methods 81: 31-42; Coteet al. (1983) Proc. Natl. Acad. Sci. 80: 2026-2030; and Cole et al.(1984) Mol. Cell Biol. 62: 109-120.)

Various methods such as Scatchard analysis in conjunction withradioimmunoassay techniques may be used to assess the affinity ofantibodies for an antigen. Affinity is expressed as an associationconstant, K_(a), which is defined as the molar concentration ofantigen-antibody complex divided by the molar concentrations of freeantigen and free antibody under equilibrium conditions. The K_(a)determined for a preparation of polyclonal antibodies, which areheterogeneous in their affinities for multiple antigen epitopes,represents the average affinity, or avidity, of the antibodies for anantigen. The K_(a) determined for a preparation of monoclonalantibodies, which are monospecific for a particular antigen epitope,represents a true measure of affinity. High-affinity antibodypreparations with K_(a) ranging from about 10⁹ to 10¹² l/mole arepreferred for use in immunoassays in which the antigen-antibody complexmust withstand rigorous manipulations. Low-affinity antibodypreparations with K_(a) ranging from about 10⁶ to 10⁷ l/mole arepreferred for use in immunopurification and similar procedures whichultimately require dissociation of antigen, preferably in active form,from the antibody. (See Catty (1988) Antibodies, Volume I: A PracticalApproach, IRL Press, Washington, D.C.; and Liddell and Cryer (1991) APractical Guide to Monoclonal Antibodies, John Wiley & Sons, New York,N.Y.)

Metal nanostructures are currently studied for a wide variety ofbiomedical applications including contrast imaging, ultrasonic imaging,thermal destruction of specific cancer cells, and laser tissue welding.All applications of this type rely on the optical and physicalproperties associated with metal nanoparticles, nominally of gold. Muchof this work has focused on gold nanoshells due to their near IR opticalabsorption where tissue transmission is at its peak, making in-vivoapplications feasible.

One of the most exciting of these applications is thermal destruction ofcancer cells. The nanostructures are selectively attached to cancercells in a tumor by a passive mechanism that has been termed an“enhanced permeability and retention effect”. The tumor mass is thenilluminated with near IR laser light which passes harmlessly through thetissue, but is absorbed strongly by the aggregates, causing them to heatdrastically, killing only the cancerous cells. (See O'Neal et al.,(2004) Cancer Lett. 209: 171-176, herein incorporated by reference inits entirety.) This technology has been utilized with gold-silicananoshells further comprising “stealthing” polymers, such aspoly(ethyleneglycol) and derives thereof, or liposomes; however this canbe done better with HGNs of the present invention.

The nanostructures disclosed herein can be formed and shaped into adesired shape, such as a sphere, a cylinder, a rod, a rod, a cone, apyramid, or other shape, not limited to regular shapes, and can bedeposited upon a substrate at a desired density using means well knownto those of skill in the art. (See, for example, Fan et al., (2005) J.Vac. Sci. Technol. 8: 947-953; Chaney et al., (2005) Appl. Phys. Lett.87: pub. no. 031908.)

Nearly monodisperse HGNs of tunable interior and exterior diameter havebeen synthesized by sacrificial galvanic replacement of cobaltnanoparticles. We have been able to control the position of the surfaceplasmon band between 550 and 820 nm by carefully controlling particlesize and wall thickness. Cobalt particle size, the sacrificial templatethat controls the resulting HGN size, is tunable by simultaneouslychanging the concentration of sodium borohydride and sodium citrate, thereductant and capping agent respectively. This varies from allpreviously reported aqueous syntheses of cobalt particles. We also showthat by controlling the addition of gold carefully the thickness of thegold shell can be varied. These HGNs have been further demonstrated tobe excellent SERS substrates in terms of spectral consistency. They arepromising for chemical and biological sensing applications, particularlythose requiring near IR absorption.

Effects of Oxygen on HGN Formation.

Cobalt is extremely sensitive to oxygen, especially in aqueous solution.If the solution is not properly de-oxygenated, or if air is allowed toenter the reaction vessel the results can be disastrous. While it isstill possible to perform the reduction of gold salt on partiallyoxidized cobalt particles, it produces very poor results. The physicalresult of this is shown in FIG. 19. While the oxidized cobalt willdissolve in the solution, it does not oxidize homogeneously whichresults in malformed HGNs. Optically, this has extremely deleteriousresults greatly broadening the absorption band due to the random natureof the oxidation. When solutions are badly oxidized, the percentage ofthese types of particles tends to increase.

EXAMPLES

The invention will be more readily understood by reference to thefollowing examples, which are included merely for purposes ofillustration of certain aspects and embodiments of the present inventionand not as limitations.

Example I Synthesis of Hollow Gold Nanospheres

HGNs were synthesized by first producing cobalt nanoparticles astemplates. 100 ml of 18 MΩ water, 500 μl of 0.1 M aqueous sodium citrate(Aldrich), and 100 μl of 0.4 M aqueous CoCl₂ (Aldrich) was degasses withnitrogen for 1 hour in a well sealed three neck flask. To this, 300 μlof a freshly prepared 1 M aqueous sodium borohydride (Aldrich) solutionwas added quickly. Hydrogen gas begins to form immediately and thesolution turns from colourless to brown. The solution is allowed to stirunder nitrogen for an additional 45 minutes to allow the sodiumborohydride to completely react. While maintaining nitrogen flow, a 0.1M aqueous chloroauric acid (Sigma-Aldrich, St. Louis, Mo.) solution isadded in 50 μl aliquots to a final volume of 500 μl. The solutionchanges from brown, to red-purple, and is finally a deep blue color.Silver particles were synthesized by the method of Lee and Meisel (Lee,P. C.; Meisel, D., J. Phys. Chem. 1982, 86, 3391-3395).

Example II Synthesis of Cobalt Nanoparticles

Cobalt nanoparticles were synthesized with the utmost attention paid tocleanliness and exclusion of air. All glassware was cleaned with alconoxglassware detergent, then aquaregia to ensure the removal of alladsorbates, and then washed repeatedly with ultra-pure water. To ensurecompletely air free solutions, all solutions were vacuumed on a Schlinkline until gas evolution ceased, then bubbled with ultra-pure argon forten minutes. This process was repeated twice to remove as much oxygen aspossible from the reaction vessel.

Fast addition of cobalt chloride. 100 ml of water was placed into athree neck flask with 100-800 μl of a 0.1 M solution of sodium citrateor citric acid and deairated. To this, 100-800 μl of a freshly made 1Msodium borohydride solution was added. With rapid magnetic stirring, 100μl of a 0.4 M-0.6 M cobalt chloride solution was added. Hydrogenimmediately evolves and the solution changes from pale pink tobrown/gray indicating the reduction of Co (II) into cobaltnanoparticles. This solution was allowed to react for between 15 and 60minutes (under constant argon flow) depending on sodium borohydrideconcentration until hydrogen stopped evolving, indicating completehydrolysis of the reductant. The addition of sodium borohydride andcobalt chloride was also performed in reverse order.

Slow addition of cobalt chloride. 75 ml of water was placed in a 500 mlthree neck flask with 400 μl of a 0.1 M solution of sodium citrate. 25ml of water with 100 μl of 0.4 M cobalt chloride was placed in a 250 mlthree neck flask. These two solutions were deairated. To the 500 mlthree neck flask, 300 μl -400 μl of a freshly prepared 1M sodiumborohydride solution was added. Using a cannula and argon gas topressurize the 250 ml flask, the cobalt chloride solution was addeddropwise at approximately 10 ml/minute. During this addition, thesolution slowly changes from colorless to brown/gray signifying cobaltparticle formation. This solution was allowed to react for 25 minutes tocompletely hydrolyze the sodium borohydride.

Example III Gold Shell Growth

Due to the ease with which sodium borohydride is able to reduce the goldsalt it is imperative that it be completely hydrolyzed beforeintroducing gold. The presence of sodium borohydride is monitored byhalting stirring and inspecting the solution for bubbles indicating thecontinuing hydrolysis of the reductant. It is only when bubbling hasceased completely that gold may be added.

High concentration addition. Upon insuring complete hydrolysis of thesodium borohydride the flow of argon is increased and a 0.1 M solutionof chloroauric acid is added at 50 μl/addition to a total volume between150 μl and 450 μl. Between each addition 30 to 60 seconds are allowed topass to ensure complete mixing. Upon completion of gold addition, theargon flow is stopped and the vessel is opened to ambient conditionsunder rapid stirring to oxidize any remaining cobalt metal left insolution.

Low concentration addition (retaining Co at core). Using a cannula, 30ml of the sodium borohydride free cobalt nanoparticle solution istransferred to an argon-purged graduated cylinder. This is then rapidlyadded to a vortexing 10 ml solution of chloroauric acid. The goldsolution contains between 20-60 μl of cloroauric acid diluted to 10 ml.To retain the cobalt core this solution may be kept under argon flow,however, by exposing the solution to air the cobalt is completelyoxidized leaving only water and dissolved salts at the core of the HGN.Samples with remaining cobalt cores retain a brown color, while oxidizedsamples change to between purple and green colored depending on amountof gold added and size of the particle.

Example IV Single Particle SERS/Luminescence and Bulk SERS

Single particle SERS and Rayleigh scattering were performed on a homebuilt confocal microscope system described previously (Schwartzberg, A.M., Grant, C. D., Wolcott, A., Talley, C. E., Huser, T. R., Bogomolni,R., and Zhang, J. Z., J. Phys. Chem. B 2004, 108, 19191-19197.) with theaddition of transmitted light dark field illumination (NA 1 to 1.4). ForSERS experiments and imaging, a Zeiss Apochromat 100X, 1.4 NA oilemersion objective was used. Typically the sample was integrated for 30seconds with a total power of 100 mW from a helium-neon laser (632.8 nm,Melles Griot). Rayleigh scattering experiments were performed with aZeiss Apochromat 100X, 0.7 NA oil emersion objective.

Samples for SERS and Rayleigh scattering were prepared by immobilizingthe particles on glass coverslips withtrimethoxy-[3-(methylamino)propyl]silane (APS) (Aldrich). Coverslipswere cleaned prior to the silanization step by sonication in a 2%solution of Hellmanex, followed by 18 MΩ water. They were then submergedin 5 mM aqueous solution of APS to deposit the tethering molecules.After one to two minutes, the coverslips were rinsed with water, driedunder nitrogen, and 40 μl of the as prepared particle solution wasplaced on one surface. After several seconds exposed to the solution, itwas rinsed with water then blown dry with nitrogen. For samples preparedfor SERS studies, the HGNs coated surface was then treated with MBA byapplying 40 μl of a 1 mM ethanoic solution for 60 seconds. The samplewas then rinsed with ethanol and dried under nitrogen.

While questions remain about the nature of the enhancement at the HGNssurface, it is the consistency of their optical response that ultimatelydetermines their reproducibility in sensor applications. To characterizethe optical response, we began by examining the Rayleigh scatteringspectra of many individual HGNs (FIG. 8, red trace, middle) and theensemble average solution absorption (FIG. 8, black trace, top), bothare indicative of plasmon resonance position and structure. Similar toother shell structures, a single peak is observed, nominally indicatingnon-aggregated HGNs (Nehl, C. L., Grady, N. K., Goodrich, G. P., Tam,F.; Halas, N. J., and Hafner, J. H., Nano Lett. 2004, 4, 2355-2359). Thespectral width of the individual particle is not significantly differentfrom that of the ensemble averaged solution, which is a result of thesample consistency. The shift of the peak position in the scatteringspectrum as compared to that of the ensemble average solution absorptionspectrum is due to a change in refractive index of the HGNs surroundingfrom air to water. This is known to affect the plasmon resonance ofparticles of this type. (See Nehl, C. L. et al., (2004) supra; Grady, N.K., Goodrich, G. P., Tam, F., Halas, N. J., and Hafner, J. H. NanoLett., 2004, 4, 2355-2359. Sun, Y. G. and Xia, Y. N., Anal. Chem. 2002,74, 5297-5305.)

The Rayleigh scattering spectra of 100 HGNs were taken and compiled(FIG. 8, bottom) into a histogram showing an average maximum scatteringintensity (λ_(max)) of 621±10.6 nm. While there is someparticle-to-particle variation it should not have an overly strongeffect on the SERS response of the particles as the shift is less than10% of the homogeneous line width. Any shift should have a minimaleffect on the absorption cross-section at the excitation or Ramanscattering wavelengths. This is clearly not the case with silveraggregates such as those shown in the inset of FIG. 8. The scattering ofindividual silver aggregates shifts drastically, by hundreds ofnanometres, and many show multiple peaks.

The homogeneous scattering properties of the HGNs suggest a consistentSERS response. To assess this, we compared the spectra of 150 HGNs and150 standard silver particles coated with the same model analyte,4-mercaptobenzoic acid (MBA). Representative SERS spectra of MBA boundto HGNs (red trace, top) and solid silver particles (blue trace, bottom)are shown in FIG. 4. The histograms in the inset show the statisticalrepresentation of the signal homogeneity of the silver (blue) and HGNssamples (red). To compare the two samples without the influence ofabsolute intensity fluctuations, two peaks were chosen to normalize theresults. Peaks at 1070 cm⁻¹ and 1590 cm⁻¹, both ring breathing modes,were chosen because these are the most intense peaks. Also, they arespaced 500 cm⁻¹ apart where any variation due to plasmon shift shouldbecome readily apparent. From the histograms in the inset of FIG. 4, itis clear that the HGNs (red) have a significantly narrower distributionthan the silver nanoparticles (blue). All peak ratios of the 150 HGNsfall within 0.9 and 1.1, representing statistical distribution of 5% bystandard deviation, while the ratios for silver particles range from 0.5to 1.7, or 45%. This is nearly a tenfold increase in consistency,demonstrating that these HGNs are a clear and significant improvementover the silver nanoparticle aggregates previously reported.

Considering the importance of probe size, especially for intracellularsensing, it is imperative to confirm that single HGNs are sufficient togenerate the observed SERS spectra, and not merely HGNs aggregates. Allsingle particle experiments were performed with this in mind. Byimmobilizing particles to the surface of glass coverslips while takingsteps to avoid aggregation we hope to minimize signal due to aggregatedHGNs. To prove that enhancement can originate from individual HGNs,however, a more detailed and involved study was required. Singleparticles treated with the sample analyte MBA on indexed TEM grids wereco-located by TEM and confocal Raman imaging. An area was located inwhich SERS was observed, while no aggregates were present within severalhundred microns (FIG. 9). The diffraction limited focal area of thelaser is indicated by the red circle, approximately 350 nm. The strongbackground of this spectrum is due to fluorescence of the holy carbonfilm on the TEM grid. While this fluorescence affects the appearance ofthe overall spectrum, it has little effect on the SERS spectrum of MBAthat contains characteristic peaks, e.g. at 1070 cm⁻¹ and 1590 cm⁻¹.This is experiment clearly demonstrates that SERS can and does originatefrom individual hollow nanostructures.

The probe molecule used, MBA, was chosen for its utility as a modelsystem in SERS pH sensing. SERS has the potential to become a valuablealternative approach to intracellular sensing compared to fluorescentdyes, because of its high sensitivity and molecular specificity. Evenwith resonant probe molecules, SERS provides robust signals that are notprone to rapid photodecomposition. In the following, we demonstrate thatisolated functional gold nanostructures provide a highly consistent andreproducible SERS response for pH detection with a direct comparison tothe aggregated silver colloids presented in earlier work (Talley, C. E.,Jusinski, L., Hollars, C. W., Lane, S. M., and Huser, T., Anal. Chem.2004, 76, 7064-7068). The response of MBA coated HGNs was taken at 7different pH points with 20-30 particles sampled individually for eachdata point. The results are shown in FIG. 10 and represent a pHcalibration curve. In FIG. 10, the SERS intensity ratio between the 1430cm⁻¹ peak, due to COO⁻ stretching mode and most sensitive to pH changes,and the 1590 cm⁻¹ ring breathing mode, which is insensitive to pH, isgraphed as a function of different bulk pH. Error bars in the intensityratio correspond to the standard deviation of each measurement andincrease with signal intensity, as error is a function of enhancementvariability and will linearly increase as the measured signal becomeslarger. The percentage error is relatively constant through the entirepH range at an average of 9.1±2.4%. In a direct comparison to previouswork, however, it is clear that for pH sensing applications, the HGNsare a significantly more precise probe than aggregated silvernanoparticles which have an average error of 104.5±71.6% (Talley, C. E.et al., (2004) supra); Jusinski, L., Hollars, C. W., Lane, S. M., andHuser, T., Anal. Chem. 2004, 76, 7064-7068). Again, this is a ten-foldimprovement over the solid silver particle system.

The most important feature to note in FIG. 10 is the narrow distributionof relative intensities represented by the error bars at each measuredpH value. The pH resolution is dependent on signal homogeneity of theprobe. With nanoparticle aggregates, resolution was limited to about 1pH unit due to the large variability resulting from the aggregatedstructures. With increased homogeneity of the HGNs, however, theresolution is now increased to 0.5 pH units or less, effectivelydoubling the sensitivity of the probe undoubtedly due to samplehomogeneity and the ability to attain SERS from individual particles. Inaddition to this improvement in pH resolution, the HGNs are sensitive toa much broader pH range. While silver substrates yield a sensing regionfrom ˜pH 6.5-pH 8, these HGNs are responsive from ˜pH 3.5-pH 9. Thereason for this wider pH sensitivity is not immediately apparent,however, it is important to recognize that with increasing particlehomogeneity, the packing of the MBA molecules at the particle surfacewill become more uniform. This will result in stronger molecule-moleculeinteractions that may have the effect of partially shielding some of theMBA from the bulk solution. This can shift the kinetics of theprotonation or deprotonation process of the acid group, effectivelyexpanding the window of sensitivity. By increasing the active range,this HGN-based probe is sensitive at most biologically relevant pHranges.

Example V Transmission Electron Microscopy (TEM)

Low resolution TEM measurements were performed on a JEOL modelJEM-1200EX microscope and High resolution TEM was performed on a PhilipsCM300-FEG at the National Center for Electron Microscopy at LawrenceBerkeley National Laboratory.

Absorption measurements were taken on a HP 89532A spectrometer. Allspectra were fit with Igor Pro 5.0 using a lorentzian function with chisquare values less than 0.1. Particles were sized with imageJ imageprocessing software (Abramoff, M. D., Magelhaes, P. J., and Ram, S. J.,Biophotonics Internat. 2004, 11, 36).

The homogeneity of the HGN samples is demonstrated in FIG. 6, whichshows a representative low resolution transmission electron micrograph(TEM) of HGNs. The HGNs have an average diameter of 30 nm with acoefficient of variation of 14%. The relatively narrow size distributionis a reflection of the cobalt seed particles from which the HGNs weregrown. The high resolution TEM of an individual HGN, shown in FIG. 7illustrates the polycrystalline, and uniform nature of the HGNs.Twinning of the lattice planes confirms that the shell is comprised ofnanocrystals that have been fused together upon growth. Due to thenature of wall growth, from the outside in, and the flow of oxidizedcobalt out of, and gold ions into the HGNs, it is likely that pinholesin the wall will remain. Computationally, it has been shown thatpinholes in hollow particle structures concentrate the evanescent fieldthat results from the excitation of surface plasmons (Hao, E., Li, S.Y., Bailey, R. C., Zou, S. L., Schatz, G. C., and Hupp, J. T., J. Phys.Chem. B 2004, 108, 1224-1229). They are, however, difficult to detect byTEM measurement. This may be a factor important to SERS enhancement andwill be explored in depth in future works.

Example VI Effect of Cobalt Chloride, Sodium Borohydride and SodiumCitrate Concentration on Particle Size

The goal of this study was to gain control of the cobalt particle sizeby aqueous solution chemical methods. Previous work on this system byLiang et al. focused more on the thickness of the shell to control itsoptical properties (Liang, H. P., Wan, L. J., Bai, C. L., and Jiang, L.J., Phys. Chem. B 2005, 109, 7795). While their work produced excellentresults, further tunability is necessary to make the system as useful aspossible. Initial attempts to reproduce the work of Liang et al. did notyield satisfactory results. The particles obtained were inhomogeneousand significantly smaller than the 60 nm reported. In fact, using asclose to precisely the same synthesis as possible, ˜25 nm cobaltparticles were obtained, however, with their method of gold additiononly inhomogeneous, gray solutions were observed. Upon determining animproved method of gold addition, this yielded excellent results forsingle particle SERS probes. However, there are many applications thatmay benefit from larger particle size and further red-shiftedabsorption, including SERS.

The other guiding hand in this work was provided by Kobayashi et al.,who first reported this cobalt particle synthesis, but proceeded to capthe particles with silica shells to protect them from oxygen (Kobayashi,Y., Horie, M., Konno, M., Rodriguez-Gonzalez, B., and Liz-Marzan, L. M.,J. Phys. Chem. B 2003, 107, 7420). Kobayashi et al. found that ascitrate concentration was reduced, particle size increased. This isconsistent with colloidal gold and silver syntheses and is not anunreasonable claim. For this application however, their trend did nothold true. A significant difference between this work and that ofKobayashi et al. is the time at which the reaction could be halted. Intheir work, for large cobalt particles, they were forced to add thesilica growth reagents almost immediately upon reduction of the cobaltsalt. Any delay at low citrate concentration and the solutions wouldbecome unstable and flocculate. In this work however, if the goldsolution is added too quickly, it is immediately reduced by theremaining sodium borohydride instead of the cobalt particles. This leadsto an unfortunate mess of nanoparticles. To achieve optimal particlegrowth a significant amount of time must pass in order to allow thesodium borohydride to completely hydrolyze before the gold can be added.

This being said, it is also important to note that even at relativelyhigh concentrations of citrate where the particles are still stableafter some time, there is little change in particle size by merelyaltering the citrate concentration. There may be a relatively simpleexplanation for this observation. Because the particle stability isdirectly related to the concentration of citrate there may have been anaggregation affect responsible for the size increase observedpreviously. As citrate concentration is reduced, we have observed thatthe rate of aggregation increased. Therefore, when capping the particlesimmediately after reduction, they are likely halting the aggregation atdifferent stages depending on citrate concentration. When concentrationis low, a larger aggregate will be formed before the silica canstabilize it, at high concentration a smaller aggregate will be present.This may be responsible for the lack of crystalline structure in the assynthesized particles. By sintering them at high temperature, they arelikely fused into one crystalline particle.

Why then, does citrate not affect particle size as strongly aspreviously thought? In the case of colloidal gold, the reduction is doneby the relatively weak reductant, citrate. This reaction is slow whichallows for thermodynamic processes to control the formation of clusters.Only as many seed particles will be formed in the reaction as can bestabilized by the cappant/reductant. This means that the capping agentconcentration will have a strong affect on the number of seed particlesand hence, particle size. In the formation of cobalt particles however,a much stronger reducing agent is required. As sodium borohydride is asignificantly stronger reductant than is technically required to reducethe cobalt salt to cobalt metal, the reduction is extremely fast, takingplace in less than one minute as opposed to five to ten minutes for thereduction of gold salt by citrate. Because of this, kinetic processesdominate the formation of seed particles. The number of seeds, andtherefore the size of the resulting particle, will be more dependent onthe rate of the reduction.

The rate of reduction can be controlled in several ways. Temperatureplays a strong role in the rate of reaction, however, little change inparticle size was observed between particles synthesized at 0° C. androom temperature. A second way to alter rate is by changing the solutionpH. The reductive potential of sodium borohydride is pH dependent. It isimportant to note at this point that contrary to previous reports ofthis synthesis, we use sodium citrate instead of citric acid. This isbecause the reaction was found to be slower at the higher pH, andparticle homogeneity was superior in the neutral solution. Higher andlower pH was also attempted by adjusting with HCl and NaOH. Thesesolutions, however, were unstable and immediately crashed out. This ismost likely due to the presence of excess ions, especially Cl⁻ which hasa strong disrupting effect on aqueous colloidal capping. Finally,altering the concentration of reductant was used to change reactionrate. This was found to be the best method of controlling particle sizewithout drastically decreasing particle homogeneity.

By decreasing the amount of sodium borohydride present, the reactiontime is increased substantially. This produces larger particles thatremain stable in solution. Table 1 shows the result of varying sodiumborohydride concentration by one quarter. The particle size is increasedby approximately 40%, however, this is the practical limit of sizetunability by this method. Lower concentrations produce incrediblyinhomogeneous results that are often unstable. In order to form largerparticles we must also alter the sodium citrate concentration.

TABLE 1 Particle size is dependent upon sodium borohydride concentrationVolume Volume Volume 0.4M 0.1M 1M Particle CoCl₂ Citrate NaBH₄ Size (μl)(μl) (μl) (nm) 100 400 400 31 ± 2 100 400 100 44 ± 5All reactions were performed in 100 ml water. All particle sizes aredetermined by examining the resulting gold particles. Reported sizes arein diameter.

While the sodium borohydride reduction of metal salts is largelykinetics driven, there are still some thermodynamic-type processescontrolling particle size. This is especially true as the concentrationof reductant is decreased and the reaction is slowed. The reaction isnow substantially more thermodynamically controlled, making thevariation in capping agent concentration more effective in controllingparticle size. By decreasing both NaBH₄ and citrate concentration weobserved a drastic increase in particle size, this is shown in the 3Dplot in FIG. 11. The trend appears to be linear, at least within theconcentrations shown here. At lower concentrations the particle sizescould be substantially larger, however, because they crash out ofsolution almost immediately this is not something we could test. Wepresent this as a general method of tuning the size of cobaltnanoparticles. Using this plot, it is possible to predict roughly whatthe final particle size will be at a given sodium borohydride and sodiumcitrate concentration.

Example VII The Influence of the Rate of Addition and Concentration ofCoCl₂ on Particle Homogeneity

To increase particle homogeneity and size, a slow addition of lowconcentration cobalt salt was attempted. It was thought that this wouldartificially slow the rate of reaction. This, however, was not the case,as is shown in Table 2.

TABLE 2 Rate of addition and concentration of cobalt salt influencesparticle size. Rate of CoCl₂ Volume Particle CoCl₂ Concentration 0.1MCitrate Volume 1M Size Addition (M) (μl) NaBH₄ (μl) (nm) Fast 0.4 400400 28 ± 2 Slow 0.4 400 400 31 ± 6 (diluted) Fast 0.5 400 400 50 ± 5

All particle sizes are in diameters. The cobalt chloride solution usedfor the slow addition is diluted to 25 ml with water.

While slightly larger particles were achieved, the coefficient ofvariation increases from 7% to 18%. This is clearly not an advantageousmethod of controlling particle size. The reason for this great increasein variation is due to the continual formation of seed particles as thecobalt is added. When examining the particles it is obvious that someseeds are formed initially and result in very large particles, whileothers are formed throughout the addition and lead to small particles.This is clear in FIG. 12 that shows histograms of particle size fromslow and fast addition of cobalt. Not only does this exemplify theinhomogeneity of the slow addition sample, it also shows the asymmetricformation of particles. While the fast addition yields a nice, evensample, the slow addition yields a curve broadened and asymmetricallyshifted by the presence of large particles formed early in the cobaltaddition. This is clearly not the way to increase particle size. Byincreasing the concentration of cobalt while maintaining volume,however, we have found that particle size changes drastically withoutexcessively broadening particle distribution, this is also shown inTable 2. While higher concentrations of cobalt seem to induceflocculation, it may be possible to better control this with carefulchanges in citrate concentration.

Example VIII Formation of Gold Shells

Along with the tunability of cobalt particle sizes we have been able toproduce a wide variety of sizes of the HGNs as shown in FIG. 13. Theseare representative TEM images of the HGNs at different sizes. FIG. 13Ais a high resolution TEM of a 30 nm particle, the lattice fringes ofgold are clearly defined and show that these particles arepoly-crystalline with large single crystalline areas. FIGS. 13B-F showthe tunability of the samples, from 70 nm to 28 nm. The largest particlesample in FIG. 13B clearly demonstrates the inhomogeneity that seems tobe inherent at larger sizes.

Forming the gold shell seems to be an extremely simple matter at firstglance, however, under closer inspection it becomes clear that there aremany parameters that must be carefully controlled in order to form highquality samples. As mentioned above, attempting to recreate the previousworks did not result in good samples. Another method was needed to makehomogeneous samples of high optical and structural quality like thoseshown in FIG. 13.

High concentration gold addition. The general consensus on homogeneousnanoparticle formation is that a low concentration of reagents yieldsthe best results. It is important to remember, however, that in theaddition of gold here, we are not forming a normal colloidalnanoparticle system. All that determines particle size and shape is thesacrificial template. For this reason the high concentration addition ofgold should not necessarily produce poor results. After many attempts,it was found that by adding high concentration (0.1 M) gold salt insmall volumes yielded excellent results. Adding the gold all at oncegave poor results, as did adding the solution dropwise. By usingapproximately 50 μl per addition over five to eight additions,spectrally narrow, highly concentrated samples were achieved.

The explanation for this is a fairly simple one: it is a matter ofmixing. The reaction of gold salt with the cobalt particle is very fast,happening almost instantaneously upon the addition of the gold. There isalso a secondary shell mediated growth that takes place on a slightlylonger time scale, where free citrate in solution will reduce excessgold salt onto the formed shells. This can result in significantlythicker shells when too much gold is added. When a small amount of goldis introduced to the stirred solution, all particles at the site of theaddition will immediately be oxidized completely in the presence of suchhigh concentration gold. If there is excess gold at this site, it willdiffuse through the solution being reduced onto the cobalt particlesuntil there is no more gold. If the volume of gold solution is too low,i.e. dropwise, the immediate impact will be relatively small but due tothe small size of the droplet it will dilute quickly. As the golddilutes into the water, less and less will be reduced onto the cobalt,resulting in a gradient of shell thicknesses. Thickest at the site ofaddition and thinner shells moving away from the concentration center.This leads to an incongruous sample in which some shells are badlyunder-formed and some are over-grown by seed mediated growth. Anexcellent example of this over-growth is in FIG. 13C. The secondparticle from the top has some slight over-growth that looks like smallparticle stuck to the surface. When the concentration is excessive thisbecomes a much more pronounced feature of the particle.

At the other end of the addition rate scale is the all-at-once additionof the gold. This suffers similar problems to the drop-wise addition,however, there is significantly more over-growth, and less under-formedparticles. We were able to overcome this problem by using a middle ofthe road approach. By using 50 μl per addition the resulting particleswere uniform and we did not observe excessive over-growth. The choice ofthis volume was not obvious and was only discovered by experimentaltrials. This method does, however, have one major flaw. Because suchhigh concentrations are used, we were not able to readily control theshell thicknesses. In theory, if the gold is added correctly, the shellthickness should be a function of the amount of gold added. This wasachieved by using relatively large volumes of low concentration gold.

Low concentration gold addition. It was determined early on in thisstudy that using low concentrations of gold would not producesatisfactory results; however, this assessment was not entirely correct.Several factors are required for the low concentration addition of goldto work properly. The first is that the solution should be mixed verywell, as quickly as possible. If the cobalt is added to the goldsolution too slowly, most of the gold will be utilized by a small numberof particles, which will lead to poor sample homogeneity. Second, thevolume of the gold salt to which the cobalt is added must be largeenough that mixing can happen very quickly. With low volumes of gold athigher concentrations there is still a pronounced mixing problem,leading to poor samples. This is the problem we observed in reproducingthe work of Liang et al. (Liang et al. (2005) supra). While the largervolumes of gold produced reasonable results, using 5 ml or 8 ml of goldsalt gave widely varying results and consistency was a major issue.Because mixing is the biggest issue in producing consistent results, itwas hypothesized that by holding the volumes of gold and cobaltsolutions constant, a more consistent result could be obtained.

By diluting varying volumes of gold salt to 10 ml with water and addingthe cobalt as quickly as possible under rapid stirring we were able toproduce homogeneous HGN with tunable wall thicknesses, similar to thework of Liang et al. (Liang et al. (2005) supra). Shell thickness varieslinearly with gold concentration, indicating that homogeneous mixing istaking place, as shown in Table 3. These are representative values froma single sample and are consistent with all other data.

TABLE 3 Wall thickness as a function of the volume of gold salt addedVolume of Volume Volume Volume 0.1M HAuCL₄ 0.1M 0.1M 1M ParticleDilluted to CoCl₂ Citrate NaBH₄ Size Wall 10 ml (μl) (μl) (μl) (μl) (nm)Thickness 25 100 600 100 40 ± 6 6.2 ± 0.6 35 100 600 100 40 ± 6 6.9 ±0.8 60 100 600 100 40 ± 6   8 ± 0.7

Example IX Effect of Particle Size and Wall Thickness on OpticalProperties

One of the major intents of all this size tuning is the control of theoptical properties of the HGN. We have found that by varying wallthickness and particle size it is possible to tune the plasmonabsorption across much of the visible spectrum as in FIG. 4. Thesespectra are representative of many experiments and show the full rangeof tunability of this system. While the full width half max (FWHM) ofthe spectra remains relatively unchanged from 500 to 750 nm at between50 and 100 nm, the last two spectra are fairly broadened to over 200 nm.This is likely due to the formation of gold shells and rings. These areshells that have not completely formed and are likely red shifted inabsorption from the complete shells. The weak shoulder at 700 nm may bedue to the presence of complete shells, while the peak is due to therings. At this time, however, it is not possible to determine the exactaffect of the presence of the rings.

By increasing particle size at a constant wall thickness the absorptionband will red-shift as the plasmon oscillation decreases in energy. Onthe other hand, increasing wall thickness at constant particle size willblue shift the absorption band. The band shifts to higher energy becauseas the inner diameter of the HGN decreases, it takes on more solidparticle like properties. As solid gold particles at these sizes haveplasmon bands at approximately 520 nm, the absorption will always shiftin this direction as wall thickness increases. This is predicted in thework of Hao et al. and is shown experimentally here in FIG. 4 (Hao, E.,Li, S. Y., Bailey, R. C., Zou, S. L., Schatz, G. C., and Hupp, J. T., J.Phys. Chem. B 2004, 108, 1224). This 3D plot shows the effect ofparticle size and wall thickness on plasmon absorption. Representingthirteen independent experiments, the trend is clearly shown here.Because the work of Hao et al. is for particle of different sizes thanthose made here, we are not able to directly correlate their results toour data. However, we are currently working on similar calculations thatshould determine if these results match well to the theory.

Because wall thickness plays such an important role in the position ofthe plasmon absorption, it is important to understand how thiscorresponds to the amount of gold added to the solution. FIG. 5 showsthe non-normalized absorption spectra of three samples made from asingle batch of 35 nm cobalt nanoparticles. The highest concentrationsample, at 60 μl of 0.1 M gold salt added absorbs most strongly at 638nm, is the most blue shifted of the three as would be expected and has awall thickness of 7±0.8 nm. The lower concentration samples at 35 ml and25 ml are red shifted to 685 nm (wall thickness 5.6±0.6 nm) and 702 nm(wall thickness 3.7±0.6 nm) respectively. Interestingly, as the bandshifts the FWHM changes only slightly from 80 nm for the 60 μl sample,to 91 nm for the 35 μl sample to 82 nm for the 25 μl sample. This is notthe trend one might expect given the propensity of solid goldnanoparticles to broaden significantly in spectrum with increasing size.This broadening is due to the introduction of new multi-pole modes whichare non-radiative and broader in energy than the normal dipole plasmonmode (Payne, E. K., Shuford, K. L., Park, S., Schatz, G. C., and Mirkin,C. A., J. Phys. Chem. B 2006, 110, 2150; Millstone, J. E., Park, S.,Shuford, K. L., Qin, L. D., Schatz, G. C., and Mirkin, C. A., J. Am.Chem. Soc. 2005, 127, 5312). In fact, upon close examination of FIG. 13it is clear that with the exception of the last two spectra, the FWHMchanges little regardless of particle size or shell thickness. Theexplanation for this is tied to the electron mean free path in gold.Because the wall thickness is much less than this length, (˜50 nm)longer axes will dominate the plasmon oscillations and the multi-polemodes which require large particles will be minimized. Interestingly,this also explains why only one absorption band is observed for thissystem, while nanorods, which also have multiple axes of oscillation,will show two.

It may be noted that as the concentration of gold added decreases, thereis a decrease in optical density as well. This is not a matter ofparticle concentration, since 10 ml of gold is added to each sample, andthe total number of HGNs is fixed to the number of cobalt particlespresent in the original solution. This is a function of absorption crosssection of the HGNs due to the different thicknesses of gold. As thewall grows thicker it will have a larger absorption cross section.

Example X Homogeneous Line Width and Inhomogeneous Broadening

To determine if, and to what extent the absorption spectrum is broadenedby inhomogeneity in the sample, we examined the Rayleigh scatteringspectra of the HGNs. While the FWHM of the ensemble averaged solution of30±2.6 nm particles is 75 nm, the single particle FWHM is 47 nm as shownin FIG. 17. This is a broadening of 27 nm that shows that the samplesare slightly inhomogeneously broadened. This is to be expected to somepoint, but is impressively small considering how sensitive thesestructures are to variance in wall thickness and local environment(Nehl, C. L., Grady, N. K., Goodrich, G. P., Tam, F., Halas, N. J., andHafner, J. H., Nano Lett. 2004, 4, 2355; Sun, Y. G. and Xia, Y. N.,Anal. Chem. 2002, 74, 5297). The sensitivity to local environment isclear upon examination of the spectral shift between the ensembleaveraged and scattering spectra. This is a shift of 14 nm and isconsistent with all particles examined. The scattering spectra weretaken from particles immobilized on glass substrates in air while theensemble-averaged spectra were taken in aqueous solution. The refractiveindex of the imbedding medium decreases from 1.33 to 1 in going fromwater to air in these two scenarios. This substantially changes theoptical properties of the HGNs. A decrease in refractive index has beenshown to correspond to a red shift, and explains our observations here.

Example XI Surface Enhanced Raman Scattering

SERS experiments were performed on solutions of as prepared HGNs withmercaptobenzoic acid (MBA) added to a final concentration of 1 mM. Atthis concentration there was no spectral shift observed which wouldindicate aggregation, therefore we can nominally say that the resultingspectra are from non-aggregated or at least minimally aggregated. Thiswas confirmed in our previous work on SERS of single HGNs that showedthat enhancement is observable from non-aggregated HGNs (Schwartzberg,A. M., Olsen, T. Y., Huser, T. R., Zhang, J. Z., and Talley, C. E.,Anal. Chem. 2006, 78, 4732-4736). Here we show the ensemble averagedSERS spectrum of MBA in FIG. 18. In terms of enhancement, when comparedin the SERS intensity to aggregated Lee and Meisel silver particles, thestandard high enhancement SERS substrate, we achieve about 10% of thesignal. This is an excellent result for nominally non-aggregatedparticles and significantly better than many current single particlesystems.

Example XII Synthesis of Metal Nanotube

Hollow gold nanotubes were synthesized by an electroless deposition onsemi-ordered, aggregated cobalt nanoparticles. The formation of cobaltnanoparticles and the electroless deposition of gold has been reportedpreviously, however, in order to form nanotubes, reaction conditions arealtered slightly. (See Schwartzberg, A. M., Olson, T. Y., Talley, C. E.and Zhang, J. Z., J. Phys. Chem. B 110, 19935-19944 (2006); Liang, H.P., Wan, L. J., Bai, C. L. and Jiang, L., J. Phys. Chem. B 109,7795-7800 (2005).)

Briefly, 100 ml of 18 MΩ purified water with 100 μl of a 0.5 M CoCl₂aqueous solution and 600 μl of a 0.1 M aqueous sodium citrate solutionwas degassed in a round bottom three neck flask under vacuum and purgedwith nitrogen three times to ensure an oxygen free environment. To this,100 μl of a 1 M aqueous NaBH₄ solution was added under vigorous magneticstirring. Hydrogen evolution was immediate and subsequently the solutionchanged in color from light pink to light brown/gray. This cobaltnanoparticle solution was allowed to stir under nitrogen for 35-40minutes until hydrogen evolution ceased to insure that all sodiumborohydride was reacted. To form the gold nanotubes, 30 ml of the stockcobalt nanoparticle solution was added to 10 ml of rapidly mixing waterwith 25 μl of 1 mM HAuCl₄ solution. This same procedure was alsofollowed in the absence of magnetic stirring to determine the effect ofthe magnetic field on the nanotube formation. In this case manualswirling of the reaction vessel was used.

Low resolution TEM measurements were performed on a JEOL modelJEM-1200EX microscope and High resolution TEM was performed on a PhilipsCM300-FEG at the national center for electron microscopy at LawrenceBerkeley National Laboratory.

Example XIII Detection of Ab-GNP Binding Interaction Using a SecondaryAb

The effect of binding an antigen to its antibody is observed by takingthe Raman spectrum of the antibody before and after exposure to theantigen through the use of SERS. To study the applicability of thismethod, a primary antibody (SC2020, Santa Cruz Biotechnology Santa CruzCalif.) and a secondary antibody (SC1616, Santa Cruz Biotechnology SantaCruz Calif.) were used. SC2020 was obtained at a concentration of 400 1g/ml and diluted by a factor of two with 20 mM HEPES buffer (pH 7.4).This solution was mixed equal volume with a GNP solution that was alsodiluted by a factor of two with 20 mM HEPES buffer. After twenty minutesof interaction, a SERS spectrum was obtained. An equal amount of SC1616was added to the system and the SERS spectrum was obtained again. Thebinding of the secondary antibody (SC1616) to the primary antibody(SC2020) caused the SERS intensity of the secondary antibody to increaseby 20-50%. This method provides an indirect means of detecting antigensin a system.

Example XIV Detection of Tumour-Antigens in Bodily Fluids

A murine monoclonal antibody raised against the CA125 ovarian cancermarker (OC125; Bast et al., (1981) J. Clin. Invest. 68: 1331-1337; Cat.No. AB19551, AbCam Ltd., Cambridge, UK) is incubated at a finalconcentration of 100 μg/ml in HEPES buffer (pH 7.4) with GNA as preparedabove at a final concentration of 1 mg/ml for twenty minutes at ambienttemperature. The mixture is then washed four times with excess samplebuffer, then stored at 4° C. until use. A fraction is subjected to SERSto obtain baseline values.

Fluid samples from individuals with diagnosed ovarian cancer areincubated with SQD in the presence of a conjugating agent and linkermolecule for 20 minutes at ambient temperature. The mixture is washedfour times and resuspended in HEPES buffer (pH 7.4) to produce SQD-Agconjugate. A fraction is subjected to SQD luminescence to obtainbaseline values.

The SQD-Ag conjugate is added to OC125-GNA mixture in HEPES incubationmedium (pH 7.4) at ambient temperature for 8 hours. Control samples arefrom individuals without diagnosed disease or disorders. The samples arethen washed four times with incubation medium, resuspended in samplebuffer, and then divided into two fractions. One fraction is subjectedto SQD luminescence. The other fraction is subjected to SERS. Baselinevalues obtained earlier are then compared with the values obtained underexperimental conditions.

Example XV Production of Antigen Specific Antibodies

Antigen substantially purified using polyacrylamide gel electrophoresis(PAGE; see, for example, Harrington (1990) Methods Enzymol. 182:488-495) or other purification techniques is used to immunize rabbitsand to produce antibodies using standard protocols. The antigen aminoacid sequence is analyzed using DNASTAR software (DNASTAR Inc., MadisonWis.) to determine regions of high immunogenicity, and a correspondingoligopeptide is synthesized and used to raise antibodies by means knownto those of skill in the art. Methods for selection of appropriateepitopes, such as those near the C-terminus or in hydrophilic regionsare well described in the art. (See, for example, Ausubel et al. supra,chapter 11.)

Typically, the oligopeptides are 15 residues in length, and aresynthesized using an Applied Biosystems Peptide Synthesizer Model 431Ausing Fmoc-chemistry and coupled to KLH (Sigma-Aldrich, St. Louis, Mo.)by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester toincrease immunogenicity. (See, for example, Ausubel et al. supra.)Rabbits are immunized with the oligopeptide-KLH complex in completeFreund's adjuvant. Resulting antisera are tested for antipeptideactivity, for example, by binding the peptide to plastic, blocking with1% BSA, reacting with rabbit antisera, washing, and reacting withradio-iodinated goat anti-rabbit IgG. In the alternative, a non-peptideantigen is used and is conjugated to KLH.

Example XVI Purification of Naturally Occurring Antigen Using SpecificAntibodies

Naturally occurring or recombinant antigen is substantially purified byimmunoaffinity chromatography using antibodies specific for the antigen.An immunoaffinity column is constructed by covalently couplinganti-antigen antibody to an activated chromatographic resin, such asCNBr-activated Sepharose (Pharmacia & Upjohn, Kalamazoo Mich.). Afterthe coupling, the resin is blocked and washed according to themanufacturer's instructions.

Media containing antigen are passed over the immunoaffinity column, andthe column is washed under conditions that allow the preferentialabsorbance of antigen (for example, high ionic strength buffers in thepresence of detergent). The column is eluted under conditions thatdisrupt antibody/antigen binding (for example, a buffer of pH 2 to pH 3,or a high concentration of a chaotrope, such as urea or thiocyanateion), and antigen is collected.

Example XVII Identification of Molecules that Interact with Antigen

Antigen, or biologically active fragments thereof, are labeled with[¹²⁵I] Bolton-Hunter reagent. (See, for example, Bolton and Hunter(1973) Biochem. J. 133: 529-539.) Candidate molecules previously arrayedin the wells of a multi-well plate are incubated with the labeledantigen, washed, and any wells with labeled antigen complex are assayed.Data obtained using different concentrations of antigen are used tocalculate values for the number, affinity, and association of antigenwith the candidate molecules.

Those skilled in the art will appreciate that various adaptations andmodifications of the just-described embodiments can be configuredwithout departing from the scope and spirit of the invention. Othersuitable techniques and methods known in the art can be applied innumerous specific modalities by one skilled in the art and in light ofthe description of the present invention described herein. Therefore, itis to be understood that the invention can be practiced other than asspecifically described herein. The above description is intended to beillustrative, and not restrictive. Many other embodiments will beapparent to those of skill in the art upon reviewing the abovedescription. The scope of the invention should, therefore, be determinedwith reference to the appended claims, along with the full scope ofequivalents to which such claims are entitled.

1-20. (canceled)
 21. A method for detecting an analyte in a sample usingthe chemical sensor comprising a plurality of metal nanoshells (hollownanospheres), the metal nanoshells having a particle size of betweenabout 20 nm and 100 nm in diameter, the method comprising the steps of:i) providing a sample; ii) providing a semiconductor quantum dotcomprising a linker molecule (LM-SQD); iii) conjugating the analyte inthe sample with the LM-SQD thereby producing an analyte-LM-SQDconjugate; iv) providing the chemical sensor of claim 1; v) incubatingthe analyte-LM-SQD conjugate with the chemical sensor for apredetermined time period; and vi) measuring the extent of bindingbetween the analyte-LM-SQD conjugate and the chemical sensor; therebydetecting the analyte in the sample.
 22. The method of claim 21, whereinthe sample is selected from the group consisting of mammalian cells,vertebrate cells, invertebrate cells, plant cells, fungal cells, moldcells, archaeal cells, bacterial cells, viruses, and bacteriophages. 23.The method of claim 21, wherein the sample is selected from the groupconsisting of blood fluids, lymph fluids, hemolymph fluids, pulmonarysurfactant fluids, peritoneal fluids, gastric fluids, xylem fluids, andphloem fluids.
 24. The method of claim 21, wherein the sample isselected from the group consisting of fluvial fluids, marine fluids,atmospheric precipitate fluids, waste-water fluids, agricultural run-offfluids, fluids comprising hydrocarbons, fluids contaminated byhydrocarbons, aerosol fluids, aqueous fluids, and non-aqueous fluids.25. A synthetic nanotube, comprising a plurality of metal nanoshells(hollow nanospheres), the metal nanoshells having a particle size ofbetween about 20 nm and 100 nm in diameter, the synthetic nanotube beingsubstantially hollow and having mean dimensions of between about 20 nmand 100 nm in diameter and at least between about 0.1 μm and 4 μm inlength, the synthetic nanotube further comprising a plurality of metalnanoshells (hollow nanospheres) and at least one detecting molecule,wherein the synthetic nanotube comprises a surface, wherein thedetecting molecule is bound to the surface, and the metal nanoshellshaving a particle size of between about 20 nm and about 100 nm indiameter.
 26. The synthetic nanotube of claim 25 wherein the syntheticnanotube comprises a metal selected from the group consisting of gold,silver, platinum, copper, aluminum, palladium, cadmium, iridium, andrhodium.
 27. A conduit for conducting fluids, the conduit comprising thesynthetic nanotube of claim
 25. 28. An electronic conductor, theelectronic conductor comprising the synthetic nanotube of claim
 25. 29.A method for synthesizing a nanotube, the nanotube comprising acrystalline metal, the method comprising the steps of (i) combining anaqueous solution of Co²⁺ salt with an aqueous solution of citrate saltthereby forming a first mixture (ii) degassing the first mixture; (iii)purging at least once with nitrogen gas; (iv) adding an aqueous solutionof NaBH₄ thereby reducing the Co²⁺ to Co⁰, and thereby forming a secondmixture comprising Co⁰ particles, the step of adding being in thepresence of an induced magnetic field and wherein the presence of theinduced magnetic filed aligns the Co⁰ particles; (v) agitating thesecond mixture until hydrogen evolution is substantially complete; (vi)adding the second mixture comprising aligned Co⁰ particles to an aqueoussolution of Au³⁺ salt; (vii) allowing the Au³⁺ to be reduced tocrystalline Au⁰ and the Co⁰ oxidized to Co²⁺, and wherein thecrystalline Au⁰ is deposited adjacent to the aligned Co⁰ therebycreating a nanotube comprising crystalline Au⁰, the method therebysynthesizing a nanotube.
 30. The method of claim 29 wherein the nanotubecomprises a metal selected from the group consisting of gold, silver,platinum, copper, aluminum, palladium, cadmium, iridium, and rhodium.